Methods of Treating Disorders Associated with Protein Aggregation

ABSTRACT

The present invention relates to methods of treatment of clinical disorders associated with protein aggregation comprising administering, to a subject, an effective amount of an anti-protein aggregate (“APA”) compound selected from the group consisting of pimozide, fluphenazine (e.g., fluphenazine hydrochloride), tamoxifen (e.g., tamoxifen citrate), taxol, cantharidin, cantharidic acid, salts thereof and their structurally related compounds. It is based, at least in part, on the discovery that each of the aforelisted compounds were able to promote degradation of aggregated ATZ protein in a  Caenorhabditis elegans  model system. According to the invention, treatment with one or more of these APA compounds may be used to ameliorate the symptoms and signs of AT deficiency as well as other disorders marked by protein aggregation, including, but not limited to, Alzheimer&#39;s Disease, Parkinson&#39;s Disease, and Huntington&#39;s Disease.

PRIORITY

This application is a continuation of International Patent ApplicationSerial No. PCT/US2010/002898 filed Nov. 4, 2010 and claims priority toU.S. Provisional Application No. 61/258,384 filed Nov. 5, 2009, U.S.Provisional Application No. 61/382,796 filed Sep. 14, 2010, and U.S.Ser. No. 12/881,976 filed Sep. 14, 2010, the contents of each of whichare hereby incorporated by reference in their entireties herein.

GRANT INFORMATION

This invention was made with government support under grant DK079806awarded by the National Institutes of Health. The government has certainrights in the invention.

1. INTRODUCTION

The present invention relates to methods of treating clinical disordersassociated with protein aggregation comprising administering, to asubject, an effective amount of an anti-protein aggregate (“APA”)compound selected from the group consisting of pimozide, fluphenazine,tamoxifen, taxol, cantharidin, cantharidic acid, salts thereof, andtheir structurally related compounds.

2. BACKGROUND OF THE INVENTION 2.1 Disorders Associated with ProteinAggregation

A number of disorders are associated with the presence of proteinaggregates. These disorders may alternatively be referred to asdisorders of protein polymerization or of protein misfolding, but arecollectively referred to herein as disorders of protein aggregation.

The classical form of α1-antitrypsin (“AT”) deficiency is an autosomalco-dominant disorder that affects approximately 1 in 2000 live births(25). It is caused by a point mutation that alters the folding of anabundant liver-derived plasma glycoprotein during biogenesis and alsorenders it prone to aggregation (43). In addition to the formation ofinsoluble aggregates in the ER of liver cells, there is an 85-90%reduction in circulating levels of AT, the pre-dominant physiologicinhibitor of neutrophil elastase. Individuals who are homozygous for themutant allele are susceptible to premature development of chronicobstructive pulmonary disease. Pulmonary involvement is believed to becaused by a loss-of-function mechanism, as lack of AT in the lungpermits elastase to slowly destroy the pulmonary connective tissuematrix (44).

AT deficiency is the most common genetic cause of liver disease inchildren and also causes liver disease and hepatocellular carcinoma inadults. In contrast to pulmonary involvement, liver inflammation andcarcinogenesis are believed to be caused by a gain-of-toxic functionmechanism. This is most clearly demonstrated by introducing the mutanthuman ATZ allele as transgene into genetically engineered mice (45, 11).Insoluble aggregates in hepatocytes, hepatic inflammation andcarcinogenesis evolve even though the endogenous anti-elastases of thetransgenic mouse are intact.

Cohort studies from an unbiased Swedish newborn screening program haveshown that only 8-10% of the affected homozygous population developclinically significant liver disease through the first 30 years of life(26). This has led to the concept that genetic and/or environmentalmodifiers determine whether an affected homozygote is susceptible to, orprotected from, liver disease. Furthermore, it has led to considerationof two general explanations for the effects of such modifiers: variationin the function of intracellular degradative mechanisms and/or variationin the signal transduction pathways that are activated to protect thecell from protein mislocalization and/or aggregation.

Studies in this area have so far indicated that the proteasome isresponsible for degrading soluble forms of ATZ (29, 46) and thatmacroautophagy is specialized for disposal of the insolublepolymers/aggregates that accumulate in the ER (30, 47). In terms ofcellular response pathways, it is thought that accumulation of ATZactivates NFκB and autophagy but not the unfolded protein response (1,16).

Aggregation of protein is associated with a number of other disorders.Among these is Alzheimer's Disease (“AD”), a disorder which affects fourmillion people in the United States and has an incidence estimated at 1in 68 individuals. As such, AD is the most common form of age-dependentneurodegeneration. Most cases are recognized by the sporadic onset ofdementia during the seventh decade of life while the less common,mutation-linked familial cases cause dementia that is recognized by thefifth decade. AD is associated with the accumulation ofaggregation-prone peptides in the brain, especially amyloid-β (“Aβ”)peptides, but hyperphosphorylated tau proteins also contribute to thetangles and plaques that constitute the histological hallmarks of thedisease.

AD is thought to be caused by a gain-of-toxic function mechanism that istriggered by the accumulation of aggregated Aβ and tau and worsened byaging (36). Recent studies have shown that the prevalence ofautophagosomes is increased in dystrophic neurons of the AD brain, afinding that is recapitulated in mouse models of the disease (37). Mostof the evidence suggests that autophagy plays a role in disposal ofaggregated proteins that might have toxic effects on neurons (38, 39).In fact, the neuropathological effects of Aβ in a mouse model of AD wereameliorated by enhancing autophagy via overexpression of the autophagyprotein beclin 1 (39). In a study by Cohen et al., breeding of a mousemodel of AD to a mouse model with targeted disruption of the IGF-1receptor demonstrated that reduced IGF-1 signaling blunted and delayedthe toxic effect of Aβ accumulation (40). Although this could beattributed in part to sequestration of soluble Aβ oligomers into denseaggregates of lower toxicity, it is well established that IGF-1signaling inhibits autophagy and therefore that these mice would likelyhave enhanced autophagy. Thus, based on the current literature,autophagy may be increased in AD, but the load of oligomers may be toogreat to avoid toxic Aβ accumulation.

Other disorders associated with increased protein aggregates includeParkinson's Disease and Huntington's Chorea. Parkinson's Disease isassociated with the presence of protein aggregates in the form of “LewyBodies”, which contain a number of proteins including one or more ofalpha-synuclein, ubiquitin, neurofilament protein, alpha B crystallinand tau protein. Interestingly, a number of other disorders manifestedas dementia are also associated with the presence of Lewy Bodies inneurons—these include Alzheimer's Disease, Pick's Disease, corticobasalatrophy, multiple system atrophy, and so-called “dementia with LewyBodies” or “DLB”. Huntington's Chorea is associated with aggregates ofhuntingtin protein containing a mutation that results in long tracts ofpolyglutamine (“polyQ”) which result in improper protein processing andaggregate formation.

Recently, Hidvegi et al. have reported that the anti-convulsant drugcarbamazepine was able to promote the degradation of ATZ protein incells and animals manifesting the ATZ mutation, and was observed todecrease the amount of ATZ accumulated in the liver in a mouse model ofAT deficiency (41).

2.2 Drug Screening Systems

The development of small-molecule therapeutics via reverse chemicalgenetic (i.e., target-directed) screens has accelerated, in part, due tothe genome-driven discovery of new drug targets, the expansion ofnatural and synthetic combinatorial chemistry compound collections andthe development of high- and ultra high-throughput screening (HTS)technologies (58,59). Despite these advances, a lead seriespainstakingly developed in vitro may be abandoned due to the loss ofactivity or an unfavorable therapeutic index upon testing in mammaliancell cultures, vertebrate animals or phase 1 clinical trials (60, 61).Frequently, attrition of a lead series is due to unfavorable drugabsorption, distribution, metabolism, excretion or toxicity (ADMET) (62,63).

Some ADMET deficiencies can be avoided, by conducting the initial drugscreens in cells. Initially, however, cell-based screening systemssuffered from a lack of assay robustness, intensive labor and resourceutilization, and low throughput; especially in terms of dataacquisition, storage and analysis. The subsequent development ofhigh-content screening (HCS) technologies, which combines automatedfluorescence microscopy with quantitative cellular image analysis, hasconverted cell-based screening into a viable platform for HTS drugdiscovery campaigns (64). The major advantage of HCS is the simultaneousacquisition of multiple information-rich parameters (e.g., size, shape,granularity and fluorescence intensity) for each cell in culture (65).Temporal and spatial integration of these parameters facilitates theevaluation of compound effects on complex physiological processes suchas cell death activation, cell-to-cell contacts, vesicular traffickingand the translocation of fluorescent markers to different subcellularlocations (64, 65).

While HCS using cell-based assays facilitate the elimination ofcompounds that are directly cytotoxic, they are unable to identify thosethat lose their desired therapeutic effect in vivo, or demonstratedeleterious side effects on complex developmental or physiologicalprocesses, such as cellular migration or synaptic transmission,respectively. For this reason, forward chemical genetic screens (i.e.,phenotype-directed) using live animals that model human diseasephenotypes might serve as suitable alternatives to target-directedreverse chemical screens (66). Drug screens using live organisms provideseveral distinct advantages over molecular- or cell-based assays andinclude: 1) the assessment of ADMET characteristics at the earlieststages of the drug discovery process, 2) the identification of leadswithout detailed knowledge of specific disease-related targets ormolecular pathways and 3) the avoidance of ascertainment biasesassociated with targeting pathways or molecules whose involvement mayprove to be tangential to the disease process. Despite these advantages,the assimilation of live animals into drug screening protocols presentslogistical challenges. These barriers include labor- and cost-intensivedevelopment of suitable disease phenotypes; screening protocols that arelow-throughput and unamenable to statistically robust HCS-like formats;and the prohibitive consumption of compound libraries. Over the lastseveral years, however, investigators began adapting small organisms,such as Caenorhabditis elegans and Danio rerio, to HTS protocols(67-75). Taken together, these studies suggest that organisms dispensedby automated liquid-handling workstations and cultivated in microtiterplates may provide an economical alternative to molecular and cell-basedscreens. C. elegans, in particular, should be an ideal candidate forlive animal HCS campaigns, as their tissues are transparent at alldevelopmental stages, the use of fluorescent probes and tissue-specificfluorescent transgenic markers to study physiological processes in vivoare well established, fundamental cell biological processes are highlyconserved across species, and aspects of mammalian diseases can besuccessfully modeled in these invertebrates (reviewed in 76).Nonetheless, experimental variables that affect high-quality HCSprotocols, such as sample preparation, assay strategy, and imageacquisition, have yet to be optimized for any organism (77).

3. SUMMARY OF THE INVENTION

The present invention relates to methods of treatment of clinicaldisorders associated with protein aggregation comprising administering,to a subject in need of such treatment, an effective amount of ananti-protein polymer (“APA”) compound selected from compounds listed inTABLES 4 and 5 herein, and particularly selected from the groupconsisting of pimozide, fluphenazine (e.g., fluphenazine hydrochloride),tamoxifen (e.g., tamoxifen citrate), taxol, cantharidin, cantharidicacid, salts thereof and their structurally related compounds. It isbased, at least in part, on the discovery that each of the aforelistedcompounds were able to promote degradation of aggregated ATZ protein ina Caenorhabditis elegans model system and pimozide and fluphenzinehydrochloride decreased ATZ in a human cell line. According to theinvention, treatment with one or more of these APA compounds may be usedto ameliorate the symptoms and signs of AT deficiency as well as otherdisorders marked by protein aggregation, including, but not limited to,Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease.

In further non-limiting embodiments, the present invention relates tomethods and compositions for high content drug screening in C. eleganswhich may be used to identify compounds that treat disorders associatedwith protein aggregation. It is based, at least in part, on thediscovery that C. elegans, genetically modified to create a model systemfor disorders of protein aggregation, could be used, in a highthroughput screening system, to identify agents that reduce the amountof aggregated protein (in particular, ATZ protein). In preferredembodiments, the assay system of the invention utilizes an all-liquidwork-flow strategy that essentially eliminates a major bottleneck in thescreening process and fully exploits the advantages of C. elegans as aplatform for in vivo high-content and high-throughput pre-clinical drugdiscovery campaigns. According to the invention, adapting an automatedsystem that streamlines the image acquisition and data analysiscomponents to accurately define objects and detect tissue-specificchanges using fluorescent markers enables monitoring complexphysiological processes and screening for compounds that modulate theseprocesses.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-D. Overlap-extension PCR strategy for introducing introns. Tooptimize expression in C. elegans, synthetic introns (˜70 bp) wereintroduced into the ATZ cDNA. (A) Oligonucleotides primers flanked withsynthetic intronic sequences (colored) were used to amplify small (˜250bp) fragments of the ATZ cDNA. (B) PCR fragments were gel purified andadjacent fragments were incubated for 45 s at 95° C. and 68° C. for 10cycles in the presence of dNTPs and Pfu DNA polymerase to promoteannealing and extension of complementary strands. (C) Agarose gelshowing individual PCR fragments (lanes 1-5). Overlap extension products1+2 (lane 6), 4+5 (lane 7), [1+2]+3 (lane 8), [1+2+3]+[4+5] (lane 9).(D) ATZ with 4 synthetic introns (colored).

FIG. 2A-E. DIC (left) and fluorescence (right) photomicrographs oftransgenic animals. For orientation in each paired set of figures, whitearrowheads indicate corresponding basal surfaces of intestinal cells.(A) Adult worm harboring Pnhx-2::GFP shows diffuse intracellular GFPexpression within the intestinal cells. (B) and (C) Pnhx-2::sGFP andPnhx-2::sGFP::ATM transgenic animals secrete GFP into the extracellularpseudocoelomic space (asterisks). Note only background autofluorescentgranules (lysosomes) in intestinal cells, but no cytoplasmic GFP at evenhigh integration. (D) Pnhx-2::sGFP::ATZ animals accumulate ATZ“globules” within intestinal cell cytoplasm (arrowheads with dot) andfail to secrete detectable amounts of fusion protein into thepseudocoelomic space. (E) A second larval (L2) stage, Pnhx-2::sGFP::ATZanimal showing prominent intracellular inclusions of sGFP::ATZ (redarrowheads) that are comparable to those of the adult. In some animals,a second type of granule was seen occasionally (arrowheads with “x”).The subcellular location of this granule has not yet been identified.

FIG. 3A-B. Immunoblot of worm lysates after SDS-PAGE. Immunoblots wereprobed with anti-human AT (A) and anti-GFP (B) antibodies. Lane 1, N2;lane 2, Pnhx-2::sGFP; lane 3, Pnhx-2::sGFP::ATM; lane 4,Pnhx-2::sGFP::ATZ; lane 5, purified plasma AT standard.

FIG. 4A-D. Electron micrographs of ATZ globule-containing intestinalcells of transgenic worms. Cross and transverse sections of early larvalstage worms expressing sGFP::ATM (A) or sGFP::ATZ (B) transgenes.Arrowheads point to large intracellular inclusions similar to thosefound in ATZ liver. A close-up of another ATZ inclusion (C). A highermagnification of the boxed area is shown in (D). Arrowheads point toribosomes of the dilated ER. Int, intestinal lumen.

FIG. 5. ArrayScanVTi screen shot interface. Bright field and fluorescentimage of sGFP::ATZ animals in one of four fields from well D1.

FIG. 6. Detection of worm boundaries and ATZ aggregates. Objects (blueoutline (there are two bright lines at the outer boundaries of eachworm; the outer one is red (e.g. marked by an arrowhead with a dot) andthe inner one is blue (e.g. marked by an arrowhead with an “x”)) andspots (red dots, e.g. see dots indicated by white arrowheads) selectedby the ArrayScanVTi algorithms. Only these selected elements were usedfor subsequent data analysis (see FIGS. 7 and 8).

FIG. 7. Detection of fluorescent “aggregates” in N2, sGFP::ATM andsGFP::ATZ worms. Top images are merged bright field and GFP fluorescenceimages (top) were analyzed quantitatively for size and number offluorescent “aggregates” (bottom). The algorithm does not distinguishbetween GFP in the pseudcoelomic space (sGFP::ATZ animals) and GFP inactual inclusions (sGFP::ATZ). However, these GFP collections varysignificantly in size and number, which permits discrimination betweenthe transgenic lines. See FIG. 8 for analysis.

FIG. 8A-C. Comparison of aggregate number (A), total aggregate area (B)and average aggregate size (C) in N2, sGFP::ATM and sGFP::ATZ animals.Data were derived from those animals shown in FIG. 7.

FIG. 9. New improved transgenic line with mCherrry expression (marked bya white arrowhead) in the head region.

FIG. 10A-E. Global strategy for high-throughput screening of leadcompounds that alter ATZ aggregation. (A) Compounds, media and E. coliare dispensed into 96-well plates using a high-throughput robotic liquidhandler. (B) Worms are sorted based on size and GFP fluorescence andautomatically transferred into 96-well plates. (C) An automatedhigh-throughput microscopic imaging system captures images and convertsthem into numerical data that identifies changes in aggregate number,size and intensity. (D) Lead compounds are identified and furtherscrutinized to confirm positives and eliminate nuisance compounds. (E)New compounds are synthesized to develop potential therapeutics.

FIG. 11. B-score analysis of LOPAC compounds.

FIG. 12A-E. Animal (object) detection using the ArrayScan V^(TI).Thirty-five adult or mixed stage animals were dispensed into 384-wellplates, imaged and analyzed using the ArrayScan V^(TI) and SpotDetectorBioApplication. (A) A brightfield image of adult animals. (B)SpotDetector correctly identified all the worms in the field asindicated by the blue outline (e.g. marked with white arrowheads,approximately >30 worms). (C) A representative brightfield image of awell containing 36 animals with a predetermined percentage (0, 25, 50,75 and 100%) of adults sorted into a 384-well plate. (D) SpotDetectorwas optimized to identify large (L4 and adult stage) worms (blueoutline, e.g. shown by white arrowheads) and exclude smaller (L1, L2 andL3 stage) worms (orange outline, e.g. shown by arrowheads with dots).(E) Correlation between the percent of adults actually sorted per wellin d vs. the percent of adults as determined by SpotDetector. The slopeand goodness-of-fit (r²) of the linear regression were 0.72 and 0.85,respectively. The slope of the line was significantly different to I(P<0.05). Scale bar, 450 μm.

FIG. 13A-Q Automated detection and quantification of cells, tissues,subcellular protein aggregates or autophagy in individual animals. (A-J)The SpotDetector BioApplication was used to identify and quantitatedifferent types of transgene expression (left of panels) in adultanimals. The brightfield channel (left panels) was used to discriminatebetween complete adult animals (outlined in blue, e.g. indicated bywhite arrowheads) and debris or incomplete animals (outlined in orange,e.g. indicated by white arrowheads with dots), while a fluorescencechannel (colored overlays in right panels) was used to detect differenttypes of fluorescently tagged transgenes in correctly identifiedobjects. (K-N) Fluorescence images of well-fed (K) and starved (M)animals expressing the autophagy marker, mCherry::LGG-1. In well-fedanimals, mCherry::LGG-1 was diffusely cytoplasmic (K). In contrast,induction of autophagy by starvation leads to a punctate fluorescencepattern within intestinal cells, as LGG-1 is incorporated in toautophagosomes (M). (L, N) Higher magnification of the boxed areas in kand m, respectively. (O-Q) The different types of transgene expressionwere quantified by spot count (O), spot area (P) or spot intensity (Q)per animal. Spot count, spot area and spot intensity values for each ofthe transgenic lines were significantly (Student's t-test, P<0.001)different to that of N2 animals. Data derived from 10-50 wellscontaining ˜20 animals/well. Scale bars, 225 μm (A-J, K, M), 50 μm (L,N).

FIG. 14A-N.|Identification of live cells or dead animals using C.elegans. The ArrayScan V^(TI) and SpotDetector BioApplication was usedto discriminate between wild-type and toxic gain-of-function mec-4(d)mutants based on the survival of the 6 mechanosensory neurons in C.elegans. Brightfield (left), fluorescence (center) and SpotDetectorrendered (right) images are depicted for each line. (A-C, M) In N2(wild-type) animals, P_(mec-4)GFP expression was evident within 5.7±10.7touch-sensing neurons (arrowheads). (D-F, M) In the mec-4(d) mutantbackground, the number of P_(mec-4)GFP expressing neurons (arrowheads)was significantly reduced and averaged 2.0±0.7 neurons per animal. (g-i,m) No GFP-positive neurons were identified in non-transgenic, N2 worms.Data derived from minimum of 32 wells containing ˜20 animals/well.Statistical significance determined using the Student's t-test,**P<0.001. The system was then used to discriminate live from deadanimals. (J-L) Adult worms expressing the pharyngeal marker,P_(myo-2)mRFP, were incubated with various concentrations of NaAz,stained with SYTOX® Green and imaged using the ArrayScan V^(TI) (J, K).The SpotDetector BioApplication was optimized to determine thepercentage of dead animals by counting the number of SYTOX®Green-positive bodies (l) and dividing by the total number ofP_(myo-2)mRFP-positive heads detected in the GFP and TRITC fluorescencechannels, respectively. (N) Percentage of dead animals at different NaAzconcentrations as determined by visual inspection versus that determinedby SpotDetector. The slope and goodness-of-fit (r²) of the linearregression were 1.0 and 0.95, respectively. The slope of the line wasnot significantly different to 1 indicating near 1:1 correlation(P>0.95). Scale bars, 100 μm (a-i), 225 μm, (j-l).

FIG. 15A-C. Identification of animals in a mixed population using afluorescent head-marker. Thirty-six animals expressing the pharyngealmarker, P_(myo-2)mRFP were sorted into wells of 384-well plate. Thewells contained different percentages (0-100%) of L4/young, and theSpotDetector BioApplication was optimized to select this group andreject younger animals (L1, L2 and L3 stages). (A) A brightfield-mRFPcomposite image of transgenic worms at different stages expressingP_(myo-2)mRFP. (B) A SpotDetector image showing the ability todifferentiate adults (magenta overlay, e.g. white arrowheads) fromearlier staged larvae (white overlay, e.g. white arrowheads with dots)based on a combination of fluorescent spot area and intensity in thepharyngeal region. (C) Correlation between the percent of adultsactually sorted per well vs. the percent of adults as determined bySpotDetector. The slope and goodness-of-fit (r²) of the linearregression were 0.92 and 1.0, respectively. The slope of the line wasnot significantly different to 1 indicating near 1:1 correlation(P>0.05). Scale bar, 450 μm.

FIG. 16A-K.|High-content analysis of transgenic animals expressing thewild-type (ATM) and mutant (ATZ) forms of human α1-antitrypsin (AT)fused to GFP. Thirty-five young adult animals were sorted into wells ofa 385-well plate and imaged using the ArrayScan V^(TI). (A, D)Brightfield images of sGFP::ATM and sGFP::ATZ expressing transgenicanimals, respectively. (B, E) SpotDetector images of fluorescentred-heads for corresponding transgenic lines pictured in a and d,respectively. (C, F) SpotDetector images of sGFP::ATM and sGFP::ATZexpressing transgenic animals imaged in b and e, respectively. (G) Theaverage number of transgenic animals in each well was determined bycounting the number of mRFP-positive heads in channel 2 (TRITC). (H-J)The amount of sGFP::ATZ (green intracellular inclusions) accumulatingwithin the intestinal cells of transgenic animals was compared to thatof the sGFP::ATM line using the SpotDetector BioApplication to analyzethe signal detected in channel 3 (GFP). Animals expressing the mutantprotein (ATZ) were distinguished clearly from those animals expressingthe wild-type protein (ATM) whether comparing total spot count (H), area(I) or intensity (J) per animal. Number of animals analyzed 2,240 (ATM)and 2,240 (ATZ). Error bars represent SD. (K) Assay quality was assessedusing a scatter plot comparing total GFP-spot area/well (n=100 wells or3,500 animals per strain) of sGFP::ATZ animals (◯) to that of wild-typeanimals (). Solid and dotted lines indicate the mean spot area±3standard deviations from the mean, respectively. The Z′-factor for thisassay≈0.7.

FIG. 17A-K. LOPAC library screen. (A) Total spot area per animal(object), (B) z-scores and (C) B-scores from a representative screenassaying the effects of 1280 LOPAC compounds on sGFP::ATZ accumulationin transgenic animals. The x-axis represents the molecularidentification (Mol ID) number of the compound. Known autofluorescentcompounds were excluded from the plot. Selected compounds, based onrank-order were analyzed for dose-dependent responses. Well images anddose-responses were obtained for compounds that decreased ((D)cantharidin, (E) fluphenazine and (F) pimozide) or increased ((G)tyrphostin AG 879) sGFP::ATZ accumulation. In each panel (D-G), wellimages on the left and right are DMSO (control)- and drug-treatedanimals, respectively. (H-K) Higher magnification fluorescent (top) andmerged DIC (bottom) images of (H, J) DMSO- or (I, K) cantharidin-treatedanimals. Note loss of GFP::ATZ accumulation in the cantharidin treatedanimal. Scale bars, 450 μm (D-G) and 50 μm (H-K). Error bars representSEM. Number of animals used was 140 for each compound concentration and520 for the DMSO control. Significance was determined using an unpairedStudent's t-test. Asterisks indicate values that differed significantlyfrom animals treated with DMSO. *P<0.01 and **P<0.001.

FIG. 18A-E. Induction of autophagy by hit compounds. Images oftransgenic animals expressing Pnhx-2mCherry::lgg-1 treated with variouscompounds are shown. Images were acquired using a Nikon instrumentsTiEclipse widefield light microscope fitted with a 20× Plan Apochromatobjective. Images were then deconvolved using Volocity (Perkin Elmer, v5.3.2). Deconvolved z planes were then merged to a single plane.Well-fed animals treated with DMSO (A) show a diffuse mCherry expressionthroughout the intestine. In contrast, animals treated with Cantharidin(B), Fluphenazine (C) and Pimozide (D) show a markedly punctatedistribution pattern indicative of increased autophagic activity.Starved (E) animals are included as a positive control for autophagy.Scale bar, 50 pan.

FIG. 19. Dose response effect of pimozide in HCS assay.

FIG. 20. Dose response effect of fluphenazine in HCS assay.

FIG. 21. Dose response effect of fluphenazine in HCS assay.

FIG. 22. HTO/Z cells were incubated for 24 hrs in the absence orpresence of fluphenazine.

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of description and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

-   -   (i) treatment agents;    -   (ii) disorders of protein aggregation;    -   (iii) methods of treatment;    -   (iv) assay system constructs;    -   (v) assay model systems; and    -   (vi) screening assays.

5.1 Treatment Agents

Treatment agents which may be used according to the invention includethe APA compounds listed in TABLES 4 and 5 herein, and particularlypimozide, fluphenazine, tamoxifen, taxol, cantharidin, cantharidic acid,their salts (where applicable) and structurally related compounds.

5.1.1 Pimozide

Pimozide is1-[1-[4,4-bis(4-fluorophenyl)butyl]-4-piperidyl]-1,3-dihydrobenzoimidazol-2-one(IUPAC name) and has the structural formula:

Compounds structurally related to pimozide include other compounds ofthe diphenulbutyl piperidine class as well as the compounds clopimozide,penfluridol, piamperone and R28935.

5.1.2 Fluphenazine

Fluphenzine (also known, for example, as “Prolixin®” and “Permitil®”) isa drug of the piperazine subclass of phenothiazines, and specifically is44342-(Trifluoromethyl)phenothiazin-10-yl]propyl]-1-piperazineethanol,frequently available as dihydrochloride having molecular formulaC₂₂H₂₈F₃N₃OS₂HCl. The molecular structure of the dihydrochloride formis:

In addition to the hydrochloride, fluphenazine is also commerciallyavailable as fluphenazine decanoate. Complexes of fluphenazine withcompounds other than hydrochloride and decanoate fall within the scopeof the invention.

Compounds structurally related to fluphenazine include other members ofthe piperazine subclass of phenothiazines and include, but are notlimited to,2-(trifluoromethyl)-10-[3-(diethanolamino)-2-hydroxypropyl]phenothiazine,fluphenazine-4-chlorophenoxy-isobutyrate ester; compounds set forth in(102);2-nitro-10-[3-[4-(2-hydroxyethyl)-1-piperazinyl]propyl]phenothiazine;2-(2,2-dicyanoethenyl)-10-[3-[4-(2-hydroxyethyl)-1-piperazinyl[propyl]phenothiazine;2-(2-nitro-ethenyl)-10-[3-[4-(2-hydroxyethyl)-1-piperazinyl[propyl]phenothiazine and 10-[3-[4-(2-hydroxyethyl)-1-piperazinyl[propyl]phenothiazine-2-carbonitrile (103).

5.1.3 Tamoxifen

Tamoxifen (e.g., tamoxifen citrate, sold as Nolvadex® and Istubal®), is(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethylamine (e.g.,citrate) (or, in alternative nomenclature,[trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. Itsmolecular formula is C₂₆H₂₉NO (and if citrate is present, add C₆H₈O₇)and the structural formula of tamoxifen is:

Tamoxifen may optionally be administered in the form of a citrate saltor in combination with a compound other than citrate.

Compounds structurally related to tamoxifen include, but are not limitedto, tamoxifen's metabolite, 4-hydroxy tamoxifen;(Z)-4-hydroxy-N-desmethyltamoxifen (endoxifen); a nido-carborane analogof tamoxifen, boroxifen; and compounds as described in (104).

5.1.4 Taxol

Taxol, also known by the generic term paclitaxel and the trade names offormulations Taxol® and Onxal®, is5β,20-Epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one 4,10-diacetate2-benzoate 13-ester with (2R,3S)—N-benzoyl-3-phenylisoserine. Thechemical formula of taxol is C₄₇H₅₁NO₁₄ and the structural formula is(105):

Compounds structurally related to taxol include, but are not limited to,2-debenzoyl-2-(m-azidobenzoyl) paclitaxel; 2-debenzoyl-2-(phenoxyacetyl)paclitaxel; 7-benzoyl paclitaxel; N-debenzoyl-N-(phenoxyacetyl)paclitaxel (see 106); 2′-deoxypaclitaxel; 2′-methoxypaclitaxel andpaclitaxel 2′ acetate (see 107).

5.1.5 Cantharidin/Cantharidic Acid

Cantharidin is2,6-Dimethyl-4,10-dioxatricyclo-[5.2.1.02,6]decane-3,5-dione. Itschemical formula is C₁₀H₁₂O₄ and its chemical structure is:

Cantharidic acid is5′-3′2,3-dicarboxy-2,3-dimethyl-1,4-epoxycyclohexane. Its chemicalformula is C₁₀H₁₄O₅ and its structural formula is

Compounds structurally related to cantharidin and cantharidic acidinclude, but are not limited to, norcanthiridin and analogs described in(108-111).

5.2 Disorders of Protein Aggregation

Disorders of protein aggregation (also sometimes referred to in the artas disorders of protein aggregation or accumulation) that may be treatedaccording to the invention include, but are not limited to,α1-antitrypsin deficiency, Alzheimer's Disease, Parkinson's Disease,Pick's Disease, corticobasal atrophy, multiple system atrophy, Lewy BodyDisease, familial encephalopathy with neuroserpin inclusion bodies(FENIB), Huntington's Disease, amyloidosis (e.g., primary, secondary,familial, senile), prion-associated diseases (e.g., Creuzfeld-Jacobdisease, mad cow's disease), protein aggregation resulting from ischemicor traumatic brain injury (for example dementia pugilistica (chronictraumatic encephalopathy)), progressive supranuclear palsy, Lytico-Bodigdisease (Parkinson dementia complex of Guam), ganglioma, subsacutesclerosing panencephalitis, certain forms of congenital diabetes,certain forms of retinitis pigmentosa, certain forms of long QTsyndrome, hereditary hypofibrinogenemia, certain forms of osteogenesisimperfecta, certain forms of hereditary angioedema, Charcot-Marie-Toothdisease and Pelizaeus-Merzbacher leukodystrophy.

5.3 Methods of Treatment

The present invention relates to methods of treating clinical disordersassociated with protein aggregation comprising administering, to asubject in need of such treatment, an effective amount of one or moreAPA compound. Suitable APA compounds are described above and are listedin TABLES 4 and 5 herein.

A subject in need of such treatment may be a human or a non-humansubject, and may be suffering from a disorder associated with proteinaggregation or be at risk of developing such a disorder due to age,family history, or exposure to a toxic agent.

An effective amount, as that term is used herein, is an amount that (i)reduces one or more sign and/or symptom of the disorder; and/or (ii)inhibits progression of the disorder; and/or (iii) prolongs survival ofthe subject. It is this reduction in a sign and/or symptom, inhibitionof progression, or prolongation of survival which constitutes treatmentof the disorder.

Signs and symptoms of a disorder associated with protein aggregationdepend upon the particular disorder and are known to the person skilledin the art. For all disorders treated according to the invention, onesign that may be “reduced” may be the accumulation of aggregatedprotein, in which either the rate of accumulation may be slowed or (butnot necessarily) the amount of aggregated protein accumulated maystabilize or decrease.

For example, but not by way of limitation, where the disorder isAT-deficiency, signs or symptoms that may be reduced or otherwiseameliorated according to the invention include hepatitis, hepaticenlargement, hepatic fibrosis, hepatocarcinoma, impaired liver function,abdominal distension from ascites, jaundice, edema, enlarged spleen,hypersplenism, gastrointestinal bleeding, encephalopathy, renal failure,prolonged bleeding from injuries, shortness of breath, wheezing, cough,decreased serum oxygen, increased serum carbon dioxide, increased totallung capacity, decreased FEV1/FVC ratio, increased incidence ofpulmonary infection, pulmonary infection, weight loss and fatigue.Although the working example below addresses effects of ATZ accumulationon the liver additional evidence is consistent with a similar toxicfunction of ATZ in the lung, such that signs or symptoms of pulmonarydysfunction may be treated according to the invention.

As a further non-limiting example, where the disorder is AlzheimersDisease, signs or symptoms that may be reduced or otherwise amelioratedaccording to the invention include impairment of short term memory,impairment of abstract thinking, impairment of judgment, impairment oflanguage skills, and mood changes.

As a further non-limiting example, where the disorder is Parkinson'sDisease, signs or symptoms that may be reduced or otherwise amelioratedaccording to the invention include tremor, bradykinesia, rigidity,impaired speech, and dementia.

As a further non-limiting example, where the disorder is Huntington'sDisease, signs or symptoms that may be reduced or otherwise amelioratedaccording to the invention include dementia and choreoform movements.

As a further non-limiting example, where the disorder is amyloidosis,signs or symptoms that may be reduced or otherwise ameliorated accordingto the invention include thickening of the skin, rash, cardiomyopathy,congestive heart failure, cardiac arrhythmias and/or conduction defects,shortness of breath, fatigue, impaired renal function, hypothyroidism,anemia, bone damage/fracture, impaired liver function, impairedimmunity, and glossitis.

As a further non-limiting example, where the disorder is a priondisease, signs or symptoms that may be reduced or otherwise amelioratedinclude dementia and choreoform movements.

In additional non-limiting embodiment, the present invention providesfor a method of decreasing the amount of aggregated protein in a cellcomprising exposing the cell to an effective amount of one of more APAcompound. The cell may be a cell affected by a disorder of proteinaggregation, as set forth above, for example, but not by way oflimitation, a liver cell or a lung cell from a subject suffering from ATdeficiency, a neuron from a subject suffering from Alzheimer's Disease,Parkinson's Disease, Huntington's disease, a prion disease, or a cellfrom a subject suffering from any of the other aforelisted disordersassociated with protein aggregation.

The APA compound may be administered by any route of administration,including oral, intravenous, intramuscular, subcutaneous, intrathecal,intraperitoneal, intrahepatic, by inhalation, e.g., pulmonaryinhalation, etc.

For example, and not by limitation, the present invention provides for amethod of treating clinical disorders associated with proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of pimozide or a structurally relatedcompound.

In certain non-limiting embodiments of the invention, pimozide may beadministered at a total dose of between about 0.5 and 10 mg/day, orbetween about 0.5 and 1 mg/day, or between about 1-2 mg/day, or betweenabout 2 and 5 mg/day, or between about 2 and 8 mg/day, or less than 10mg/day. A compound that is structurally related to pimozide may beadministered at an adjusted version of the foregoing doses, where theadjustment compensates for any difference in potency between pimozideand the structurally related compound.

In certain non-limiting embodiments of the invention, the dose ofpimozide or a structurally related compound administered produces aserum concentration or cerebrospinal fluid concentration of at leastabout 5 micromolar, or between about 5 and about 10 micromolar, orbetween about 10 and about 20 micromolar, or between about 20 and about30 micromolar, or at least about 50 micromolar.

In certain non-limiting embodiments, the dose of pimozide or astructurally related compound may be administered daily, about everyother day, about twice a week, or about once a week.

For example, and not by limitation, the present invention provides for amethod of treating clinical disorders associated with proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of fluphenazine (e.g., fluphenazinehydrochloride or fluphenazine decanoate).

In certain non-limiting embodiments of the invention, fluphenazine(e.g., fluphenazine hydrochloride or fluphenazine decanoate) may beadministered at a total dose of between about 0.5 and 50 mg/day, orbetween about 0.5 and 2 mg/day, or between about 0.5 and 5 mg/day, orbetween about 2 and 10 mg/day, or between about 5 and 20 mg/day, orbetween 10 and less than about 40 mg/day, any of which doses mayoptionally be administered as a divided dose. A compound that isstructurally related to fluphenazine may be administered at an adjustedversion of the foregoing doses, where the adjustment compensates for anydifference in potency between fluphenazine and the structurally relatedcompound.

In certain non-limiting embodiments of the invention, the dose offluphenazine (e.g., fluphenazine hydrochloride or fluphenazinedecanoate) or a structurally related compound administered produces aserum concentration or cerebrospinal fluid concentration of at leastabout 5 micromolar, or between about 5 and about 10 micromolar, orbetween about 10 and about 20 micromolar, or between about 20 and about30 micromolar, or at least about 50 micromolar.

In certain non-limiting embodiments, the dose of fluphenazine (e.g.,fluphenazine hydrochloride or fluphenazine decanoate) or a structurallyrelated compound may be administered daily, about every other day, abouttwice a week, or about once a week.

For example, and not by limitation, the present invention provides for amethod of treating clinical disorders associated with proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of tamoxifen (e.g., tamoxifen citrate) ora structurally related compound.

In certain non-limiting embodiments of the invention, tamoxifen (e.g.,tamoxifen citrate) may be administered at a total dose of between about0.5 and about 50 mg/day or between about 0.5 and about 20 mg/day orbetween about 5 and about 15 mg/day or between about 10 and about 20mg/day, any of which doses may optionally be administered as a divideddose. A compound that is structurally related to tamoxifen may beadministered at an adjusted version of the foregoing doses, where theadjustment compensates for any difference in potency between tamoxifenand the structurally related compound.

In certain non-limiting embodiments of the invention, the dose oftamoxifen (e.g., tamoxifen citrate) or a structurally related compoundadministered produces a serum concentration or cerebrospinal fluidconcentration of at least about 5 micromolar, or between about 5 andabout 10 micromolar, or between about 10 and about 20 micromolar, orbetween about 20 and about 30 micromolar, or at least about 50micromolar.

In certain non-limiting embodiments, the dose of tamoxifen (e.g.,tamoxifen citrate) or a structurally related compound may beadministered daily, about every other day, about twice a week, or aboutonce a week.

For example, and not by limitation, the present invention provides for amethod of treating clinical disorders associated with proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of taxol or a structurally relatedcompound.

In certain non-limiting embodiments of the invention, taxol may beadministered at a total dose of between about 25 and about 175mg/m²/day, or between about 25 and about 50 mg/m²/day, or between about25 and about 100 mg/m²/day, or between about 25 and about 150 mg/m²/day,any of which doses may optionally be administered as a divided dose. Acompound that is structurally related to taxol may be administered at anadjusted version of the foregoing doses, where the adjustmentcompensates for any difference in potency between taxol and thestructurally related compound.

In certain non-limiting embodiments of the invention, the dose of taxolor a structurally related compound administered produces a serumconcentration or cerebrospinal fluid concentration of at least about 5micromolar, or between about 5 and about 10 micromolar, or between about10 and about 20 micromolar, or between about 20 and about 30 micromolar,or at least about 50 micromolar.

In certain non-limiting embodiments, the dose of taxol or a structurallyrelated compound may be administered daily, about every other day, abouttwice a week, about once a week, about once every two weeks, about onceevery three weeks, about once a month, about once every six weeks, aboutonce every eight weeks, about once every ten weeks, about once everytwelve weeks, about once every sixteen weeks, about once every 20 weeks,or about once every 24 weeks. Not by way of limitation, taxol ispreferably administered intravenously. To avoid a hypersensitivityreaction, it may be desirable to premedicate a subject about to receivetaxol or a structurally related compound with one or more medication,for example dexamethasone (e.g. 20 mg 12 and 6 hours prior totreatment), diphenhydramine (e.g. 50 mg IV 30-60 minutes prior totreatment), and/or cimetidine (e.g. 300 mg 30-60 minutes prior totreatment) or ranitidine (e.g., 50 mg 30-60 min prior to treatment).

For example, and not by limitation, the present invention provides for amethod of treating clinical disorders associated with proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of cantharidin, cantharidic acid, or astructurally related compound, where said effective amount does not havesubstantial toxic effects on the subject (both cantharidin andcantharidic acid are known to have toxic effects at relatively lowconcentrations, so that substantially non-toxic doses would need to bedetermined using standard pharmaceutical techniques which consider thatan anti-protein aggregate effect was achieved at a concentration of 100micromolar in the C. elegans assay). In certain non-limitingembodiments, the substantially non-toxic dose of cantharidin,cantharidic acid, or a structurally related compound may be administereddaily, about every other day, about twice a week, or about once a week.

Treatment according to any of the foregoing methods may be administeredcontinuously or for intervals interrupted by breaks.

5.4 Assay Constructs

The present invention provides for at least two types of assayconstructs: first, an aggregation-prone protein construct which encodesa protein with a tendency to aggregate (which may also be referred to asa “aggregatable protein”), the expression of which results in thegeneration of aggregated protein in C. elegans and second, a markerconstruct which comprises a marker gene that encodes a marker protein,the expression of which assists in the characterization of animals inthe assay, either by facilitating counting, developmental staging, organlocalization, or some other characterization of the worm. Saidconstructs may comprise a promoter active in C. elegans which mayoptionally confer tissue specific, location-specific, and/ordevelopmental stage-specific expression, operably linked to a nucleicacid encoding a protein with a tendency to aggregate or a marker protein(or, in non-limiting embodiments, both). Said constructs may optionallybe comprised in vectors known in the art (e.g., a plasmid, phage orvirus) or may be introduced directly into C. elegans. Said constructsmay further comprise additional elements known in the art, for example,but not limited to, one or more selection marker, a translationtermination site, etc.

In non-limiting embodiments of the invention, it may be desirable toexpress the protein with a tendency to aggregate and/or the markerprotein in a particular cell type or location in the worm. As such, itmay be desirable to use a promoter that is selectively active in saidcell type or region. C. elegans has been extensively characterized, andlists of cell-type and location specific promoters are known in the art(see, for example, C. elegans II, second edition, Cold Spring HarborMonograph Series, Vol 33, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1997), and www.wormbase.org. For example, and not by way oflimitation:

neuron-specific promoters include, but are not limited to, ace-1, acr-5,aex-3, apl-1, alt-1, cat-1, cat-2, cch-1, cdh-3, ceh-2, ceh-2, ceh-6,ceh-10, ceh-14, ceh-17, ceh-23, ceh-28, ceh-36, che-1, che-3, cfi-1,cgk-1, cha-1, end-1, cod-5, daf-1, daf-4, daf-7, daf-19, db1-1, des-2,deg-1, deg-3, del-1, eat-4, eat-16, ehs-1, egl-10, egl-17, egl-19,egl-2, egl-36, egl-5, egl-8, fax-1, flp-1, flp-1, gyp-3, flp-5, flp-6,flp-8, flp-12, flp-13, flp-15, flp-3, flr-4, gcy-10, gcy-12, gcy-32,gcy-33, gcy-5, gcy-6, gcy-7, gcy-8, ggr-1, ggr-2, ggr-3, glr-1, glr-5,glr-7, glt-1, goa-1, gpa-1, gpa-1, gpa-2, gpa-3, gpa-4, gpa-5, gpa-6,gpa-7, gpa-8, gpa-9, gpa-10, gpa-11, gpa-13, gpa-14, gpa-15, gpa-16,gpb-2, gsa-1, ham-2, her-1, ida-1, ina-1, lim-4, lim-6, lim-6, lim-7,lin-11, lin-4, lin-45, mab-18, mec-3, mec-4, mec-7, mec-8, mec-9,mec-18, mgl-1, mgl-2, mig-1, mig-13, mus-1, ncs-1, nhr-22, nhr-38,nhr-79, nmr-1, ocr-1, ocr-2, odr-1, odr-2 odr-10, odr-3, odr-3, odr-7,opt-3, osm-10, osm-3, osm-9, pag-3, pef-1, pha-1, pin-2, rab-3, ric-19,sak-1, sdf-13, sek-1, sek-2, sgs-1, snb-1, snt-1, sra-1, sra-10, sra-11,sra-6, sra-7, sra-9, srb-6, srg-2, srg-, srd-1, sre-1, srg-13, sro-1,str-1, str-2, str-3, syn-2, tab-1, tax-2, tax-4, tig-2, tph-1, ttx-3,ttx-3, unc-3, unc-4, unc-5, unc-8, unc-11, unc-17, unc-18, unc-25,unc-29, unc-30, unc-37, unc-40, unc-3, unc-47, unc-55, unc-64, unc-86,unc-97, unc-103, unc-115, unc-116, unc-119, unc-129, and vab-7promoters;

muscle-specific promoters include the hlh-1, mlc-2, myo-3, unc-54 andunc-89 promoters,

pharynx specific promoters include the ceh-22, hlh-6 and myo-2promoters; and gut-specific promoters include the nhx-2, vit-2, cpr-1,ges-1, mtl-1, mtl-2, pho-1, spl-1, vha-6 and elo-6 promoters.

Non-limiting examples of proteins with a tendency to aggregate include,but are not limited to, the human proteins: AT mutants ATZ, Siiyama andMmalton; huntingtin; synuclein; amyloid beta; neuroserpin; ubiquitin;neurofilament protein; alpha B crystallin and tau, or a non-humanequivalent thereof, or an aggregatable portion thereof. Because it isdesirable to be able to detect aggregated proteins in C. elegans, inpreferred, non-limiting embodiments of the invention the protein with atendency to aggregate is comprised in a fusion protein with a second,detectable protein, for example, but not limited to, a fluorescentprotein such as green fluorescent protein, enhanced green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, etc. Assuch, a portion of the native protein with a tendency to aggregate mayoptionally be omitted in the encoded fusion protein, and/or anadditional protein or peptide may be comprised in the fusion protein,for example to link the protein with a tendency to aggregate with thedetectable protein, provided that the tendency of the protein toaggregate is not substantially impaired (in specific non-limitingexamples, at least about 90 percent or at least about 95 percent or atleast about 98 percent of the native protein with a tendency toaggregate (examples of a “aggregatable portion”) is present in thefusion protein). Likewise, it may be desirable to truncate or otherwisemodify a native fluorescent protein to accommodate it into the fusionprotein; such modifications are within the scope of the inventionprovided that the fluorescent protein remains fluorescent.

Non-limiting examples of marker proteins are the various fluorescentproteins that are fluorescent in vivo known in the art, including, butnot limited to, green fluorescent protein, enhanced green fluorescentprotein, red fluorescent protein, yellow fluorescent protein, etc.

In particular, non-limiting embodiments of the invention, expression ofthe marker construct results in a single detectable region (or image)per worm. “A single detectable region” means that using a detectionmeans appropriate for the assay, expression of the marker results in apattern of detectable signal which can be perceived as a single region(for example, the pharyngeal region, the head region, the worm surface,or the entire worm) so that perception of that region allows for thediscrimination of one worm from another and therefore facilitatesaccurate counting of worms. In specific non-limiting embodimentsaccording to this paragraph, the marker gene is operably linked to apharynx-specific (e.g. myo-2) C. elegans promoter.

In particular, non-limiting embodiments of the invention, expression ofthe marker construct results in a defined number of detectable regions(or images) per worm. “A defined number of detectable regions” meansthat using a detection means appropriate for the assay, expression ofthe marker results in a pattern of detectable signal which can beconsistently perceived as a defined number of regions (for example, 2 or3 or 4, etc. regions), so that perception of that region allows for thediscrimination of one worm from another and therefore facilitatesaccurate counting of worms (where the number of images counted reflectsthe number of worms presented multiplied by a factor which is thedefined number of detectable regions per worm). In specific non-limitingembodiments according to this paragraph, one or more marker gene isoperably linked to one or more promoter so that the marker is expressedtwo detectable regions per animal, so that the number of worms presentmay be determined by counting the number of detectable regions anddividing by two. An analogous approach could be used to produce wormshaving three detectable regions which could be determined by countingthe regions and dividing by three, etc.

In related non-limiting embodiments, the invention provides for othermethods of defining worms and worm boundaries. For example, the presentinvention provides for a transgenic C. elegans worm expressingmyo-3::mCherry which is specifically expressed in the muscles andeffectively creates an outline of the worm. Such worms may be furtherengineered to express ATZ::GFP in any tissue and quantify ATZaggregation/polymerization within the myo-3::mCherry boundary.

The present invention further provides for worms carrying at least threetransgenes. In a specific, non-limiting embodiment, a transgenic C.elegans may be engineered to express the fusion proteins ATZ::YFP,myo-2::CFP and LGG-1::mCherry. These worms may be used to identify/studythe effect of a particular compound on ATZ disposition and autophagy,simultaneously. Similarly, we could engineer worms to expresshsp-4::mCherry as the third marker to study the “unfolded proteinresponse” (UPR). As an alternative non-limiting example, a C. elegansmay be engineered to express myo-2::mCherry (red-head marker),nhx-2::sGFP::ATZ (intestine) and lgg-1::CFP (blue marker for autophagy).In such worms, the red head (mCherry) region may be used to determinethe number of worms in the well, the green (GFP) regions may be measuredto determine the extent of ATZ aggregation/polymerization and the blue(CFP) region may be used to measure the level of autophagy.

In particular, non-limiting embodiments of the invention, in order tofacilitate expression in C. elegans, which is believed to be moreefficient in the presence of introns, where a nucleic acid encodingeither a protein with a tendency to aggregate (or aggregatable portionthereof) or a marker protein lacks introns, one or more (e.g., 1, 2, 3or 4, etc.) “synthetic” intron may be introduced (either by engineeringblunt-end restriction sites into the protein-encoding DNA (e.g., cDNA)by site-directed mutagenesis or by overlap-extension PCR (see below)).Synthetic introns are between 48-51 bp in length and include consensussplice acceptor (AGGUAAGU) and splice donor (CAGG) sequences at the 5′and 3′ ends, respectively.

Accordingly, in one set of particular, non-limiting embodiments, thepresent invention provides for an aggregated protein constructcomprising a nucleic acid encoding a protein with a tendency toaggregate (for example, but not limited to, a human protein such as ATZ,huntingtin, synuclein, amyloid beta, neuroserpin, ubiquitin,neurofilament protein, alpha B crystallin and tau, or a non-humanequivalent thereof, or an aggregatable portion thereof) optionallycomprised in a fusion protein with a detectable protein such as afluorescent protein, operably linked o a C. elegans promoter, where saidpromoter may, for example, be a neuron-specific promoter, a gut (e.g.intestinal) specific promoter, a muscle specific promoter, apharynx-specific promoter, or a tail specific promoter, and where saidnucleic acid encoding a protein with a tendency to aggregate optionallycomprises one or more synthetic intron.

In a specific, non-limiting embodiment of an aggregated proteinconstruct according to the invention which is expressed in intestinalcells of C. elegans, Pnhx-2sGFP::ATM may be generated by inserting a 4kb nhx-2 promoter fragment into HindIII/XbaI restriction sites of theexpression vector, pPD95.85. Then a KasI restriction site may beintroduced by site-directed mutagenesis into the GFP translational stopcodon. A 1.4 kb fragment containing the ATM cDNA and 3 synthetic intronsmay be cloned into the KasI site. Pnhx-2sGFP::ATZ may be generated bysite-directed mutagenesis of Pnhx-2sGFP::ATM, thereby generating theE342K (Z) mutation.

In a related specific, non-limiting embodiment, to improve ATZexpression, synthetic introns resembling those found in C. elegans maybe introduced into the ATZ cDNA using overlap-extension PCR (FIG. 1A-B).Large oligonucleotides consisting of ˜50 nucleotides of synthetic intron(carefully designed to contain appropriate 5′ and 3′ donor/acceptorsequences) and ˜22 nt sequence complementary to the ATZ coding regionmay be synthesized and used as primers to amplify small regions of theATZ cDNA (see FIG. 1A). The amplified fragments may be joined pairwiseusing overlap-extension PCR to generate larger fragments containingintronic regions (see FIGS. 1B and 1C). Once the 5 pieces are joinedtogether, the complete ATZ fragment containing all of the syntheticintrons may be amplified using primers flanked with Kas I recognitionsites (see FIG. 1D) and cloned into the expression vector pPD95.85 togenerate Pnhx-2::sGFP::ATZ.

In additional non-limiting specific embodiments of aggregated proteinconstructs, constructs encoding neuroserpin which are expressed inintestinal or neuronal cells of C. elegans are shown in TABLE 3 below.

In a specific, non-limiting embodiment of a marker construct accordingto the invention that is expressed selectively in the C. eleganspharynx, a transcriptional Pmyo-2mRFP fusion (where RFP is “RedFluorescent Protein”) construct may be constructed by subcloning themyo-2 promoter and the mRFP cDNA into the SphI/XbaI and NheI/EcoRV sitesof the canonical expression vector, pPD49.26, respectively.

5.5 Assay Model Systems

A “model system” according to the invention comprises a C. elegansadapted to serve as a model of a disorder of protein aggregation. Forexample, a model system may be a Caenorhabditis elegans carrying atransgene comprising an aggregated protein construct comprising anucleic acid encoding a protein with a tendency to aggregate (forexample, but not limited to, a human protein such as ATZ, huntingtin,synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein,alpha B crystallin and tau, or a non-human equivalent thereof, or anaggregatable portion thereof), optionally comprised in a fusion proteinwith a detectable protein such as a fluorescent protein, operably linkedo a C. elegans promoter, where said promoter may, for example, be aneuron-specific promoter, a gut (e.g. intestinal) specific promoter, amuscle specific promoter, a pharynx-specific promoter, or a tailspecific promoter, and where said nucleic acid encoding a protein with atendency to aggregate optionally comprises one or more synthetic intron,as described in the section above. In particular, non limitingembodiments, said C. elegans may further comprise an additionaltransgene comprising a marker construct comprising a marker geneoperably linked to a C. elegans promoter and encoding a marker protein,as described in the section above. Preferably, where the protein with atendency to aggregate is comprised in a fusion protein with a firstfluorescent protein and where the marker construct encodes a secondfluorescent protein, the first and second fluorescent protein are notthe same (for example, so that their fluorescent emissions aredistinguishable (for example, they may have a different wavelength)). Ina specific non-limiting embodiment of such a model system, expression ofthe marker construct results in a single or otherwise consistentlycountable detectable region (or image) per worm.

In particular, non-limiting embodiments, the present invention providesfor a model system for α1-antitrypsin deficiency comprising a C. eleganscarrying a transgene comprising an aggregated protein constructcomprising a nucleic acid encoding ATZ optionally comprised in a fusionprotein with a detectable protein such as a fluorescent protein,operably linked o a C. elegans promoter, where said promoter is a gutspecific promoter, and where said nucleic acid encoding ATZ optionallycomprises one or more synthetic intron. In particular, non limitingembodiments, said C. elegans may further comprise an additionaltransgene comprising a marker construct comprising a marker geneoperably linked to a C. elegans promoter and encoding a marker protein.

In another particular, non-limiting embodiment, the present inventionprovides for a model system for a disorder associated with proteinaggregation in neurons comprising a Caenorhabditis elegans carrying atransgene comprising an aggregated protein construct comprising anucleic acid encoding a protein with a tendency to aggregate (forexample, but not limited to, a human protein such as huntingtin,synuclein, amyloid beta, neuroserpin, ubiquitin, neurofilament protein,alpha B crystallin and tau, or a non-human equivalent thereof, or anaggregatable portion thereof), optionally comprised in a fusion proteinwith a detectable protein such as a fluorescent protein, operably linkedto a C. elegans promoter, where said promoter may, for example, be aneuron-specific promoter, a gut (e.g. intestinal) specific promoter, amuscle specific promoter, a pharynx-specific promoter, or a tailspecific promoter, and where said nucleic acid encoding a protein with atendency to aggregate optionally comprises one or more synthetic intron,as described in the section above. In particular, non limitingembodiments, said C. elegans may further comprise an additionaltransgene comprising a marker construct comprising a marker geneoperably linked to a C. elegans promoter and encoding a marker protein.As specific non-limiting examples of such embodiments, in a model systemfor AD the protein with a tendency to aggregate may be human amyloidbeta, or an aggregatable portion thereof; in a model system forParkinson's disease the protein with a tendency to aggregate may behuman synuclein, or an aggregatable portion thereof; in a model systemfor Huntington's disease the protein with a tendency to aggregate may behuman huntingtin, or an aggregatable portion thereof; in a model systemfor chronic traumatic brain injury the protein with a tendency toaggregate may be human tau protein, or an aggregatable portion thereof;and so forth.

Transgenic C. elegans may be prepared by methods known in the art,including, but not limited to, microinjection or microparticlebombardment. In a specific, non-limiting embodiment of the invention, anaggregated protein construct and/or a marker construct may be introducedby injection into the gonad of a young adult hermaphrodite worm, forexample, at a concentration of about 80 ng/μl.

5.4 Screening Assays

In particular, non-limiting embodiments, the present invention providesfor a method of determining whether a test compound has activity intreating a disorder of protein aggregation (and/or activity in reducingthe amount of protein polymer), comprising:

(i) administering said test compound to a plurality of transgenic C.elegans carrying (a) a first transgene comprising a nucleic acidencoding a human protein with a tendency to aggregate, or anaggregatable portion thereof, operably linked o a C. elegans promoter,where the expression of the human protein results in a detectableaccumulation of human protein in the C. elegans (which may bedetectable, for example, because the human protein with a tendency toaggregate, or an aggregatable portion thereof, may be comprised in afusion protein with a first fluorescent protein) and (b) a secondtransgene comprising a marker construct comprising a marker geneencoding a marker protein (e.g., a second fluorescent protein) operablylinked to a C. elegans promoter;

(ii) determining the change in the amount of human protein (oraggregatable portion thereof) associated with the administration of testcompound in the plurality of C. elegans (for example, by determining achange in the level of fluorescence associated with a first fluorescentprotein fused to the aggregatable protein, as compared to a standardvalue, or compared to the amount of protein prior to administration ofthe compound in the same population of worms, or compared to the amountof protein in a parallel control population of worms that have not beenexposed to the compound);

(iii) using the marker protein, determining the number of C. elegans insaid plurality (for example, where the marker protein generates a singleor a definite number of images per worm, counting those images as adirect method of determining the number of worms, and where the markerprotein may be a second fluorescent protein having a fluorescentdistinguishable from that of the first fluorescent protein fused to theaggregatable protein);

(iv) using the results of (ii) and (iii), determining the change in theamount of human protein per worm resulting from the administration oftest compound;

wherein, if administration of the test compound results in a significantdecrease in the amount of human protein per worm, then the test compoundis indicated to be therapeutically effective in a disorder of proteinaggregation.

The methods are preferably practiced in a high-throughput format whereat least 96 or at least 384 (or more) test compounds may be tested inparallel.

In a specific, non-limiting embodiment of the invention, a 96 well-basedassay may be performed as follows. Fifty-five young adult stage wormsmay be dispensed into 96-well plates using the COPAS BioSort (wormsorter). Worms may then be immobilized by the addition of 0.1 M sodiumazide to facilitate image capture. The plates may be placed into acomputerized high throughput plate reader, ArrayScanVTi (ThermofisherCellomics Products). ArrayScanVTi may be set up to rapidly scan wellsand capture multiple images using brightfield and fluorescenceparameters. Algorithms, as discussed below, may be used to identify andquantify defined spots (fluorescent granules) and objects (individualanimals). A typical screen shot of the ArrayScanVTi interface is shownin FIG. 5. In this case, brightfield and GFP fluorescence images weretaken from 4 different fields within each well using a 5× Carl Zeissobjective. A single field showing a brightfield and GFP fluorescenceoverlay illustrates GFP aggregates throughout the length of theintestine (FIG. 5, well D1). To quantify these GFP aggregates,algorithms were developed to first identify the objects of interest(adult worms) and quantify the number and intensity of the spots(aggregates). FIG. 6 shows exactly the elements from FIG. 5 that wereselected for data analysis. ArrayScanVTi correctly identified all adultworms in the field of view (blue outline) and excluded all eggs andother debris that would alter the analysis. Moreover, the algorithmidentified all the ATZ aggregates (FIG. 6, red spots, e.g. as indicatedby white arrowhead) within the set boundary (FIG. 6, red outline, theouter of two line boundaries at the perimeter of each worm).

To quantify the differences between the types of animals, the totalnumber of spots and the total and average spot area for multipledetection fields may be determined by the algorithm (for example, seeFIG. 8). In a preferred, specific, non-limiting embodiment of theinvention, a transgenic C. elegans that expresses an ATZ/GFP fusionprotein and carries a second transgene encoding a red fluorescentprotein (mCherry) as marker protein which is expressed only in the head(pharynx) region of the worm. The expression of a second fluorescentmarker that has a distinct expression pattern than GFP::ATZ (intestine)has several major advantages. First, the bright expression of themCherry protein significantly improves focus time and efficiency.Second, with optimized algorithms, red heads can be easily counted toobtain accurate worm number per well. Thirdly, since the pharynx is inthe same focal plane as the intestine, GFP::ATZ aggregates can be moreefficiently and accurately measured. By simply dividing the total GFPfluorescence in the well by the total number red heads, the average GFPfluorescence per worm can be determined, a capability that wasproblematic to achieve using the brightfield object identificationalgorithms.

In another specific non-limiting embodiment of the invention, a 384-wellassay may be performed as follows. A 384-well-based assay has severaladvantages over the 96-well format. First, one can screen more compoundsusing the same number of wounds needed for a 96-well plate. Second,images can be captured using the 2.5× objective. This reduces the numberof fields needed to capture the well from 16 to 1. For this assay, theArrayscan VTi may be fitted with a 0.63× coupler. The 0.63× couplerallows the capture of 100% of the well (as opposed to ˜90% using the 1×coupler) allowing one to account for all the worms on in the well,leading to a considerably smaller variance between replicate wells. Twoμl of stock (10 mM) compounds may be diluted with 98 μl of S-medium to afinal concentration of 200 μM drug in 2% DMSO and S-medium. Fifteen μlof the diluted compounds may then be transferred to 384-well platesusing a robotic liquid handler (EP3). Prior to the experiment, fifteenμl of 4×OP50/antibiotic solution may be added to each well. Using theCOPAS Biosort, 35 L4-young adult stage worms may be deposited into eachwell and allowed to incubate for 24 or 48 h at 22° C. At the end of theincubation period, worms may be immobilized by the addition of sodiumazide or levamisole to a final concentration of 12.5 mM and 4 mM,respectively. The worms may then be imaged using the high speed,automated imaging device, ArrayScan VTi. Image capture and data analysismay be performed using the Spot Detector BioApplication with algorithmsoptimized for worms. B-score statistical analyses may be performed toidentify compounds that had a significant (>2 SDs away from the mean)effect on ATZ aggregation. A summary of a typical LOPAC screen is shownin FIG. 11.

In another specific non-limiting embodiment of the invention, compoundtracking and data analysis for the primary HCS assay may be performedusing ActivityBase™ (IDBS, Guildford, UK), CytoMiner (UPDDI) softwareand visualized using Spotfire™ DecisionSite® (TIBCO Software Inc.,Somerville, Mass., USA) software, as described in 60-63. Customcalculators were written to process the HCS data and perform the z-scoreand B-score statistical analysis (64,65). As a measure of assay qualityand robustness, the Z′-factor 30 may be used. The Z′-factor may becalculated from the mean and the standard deviation of the negative andpositive control populations as follows:

Z′=1−((3×(σ_(p)+σ_(n)))/(μ_(p)−μ_(n)))

where σ is the standard deviation, μ is the mean and p and n arepositive and negative controls, respectively. Z′-factors between 0.5 and1.0 indicate the separation band (signal window) between the positiveand negative controls is wide and the assay is of excellent quality andsuitable for HTS/HCS. Z′-factors between 0 and 0.5 indicate a goodquality screen, whereas a score <0 indicates the assay is of poorquality and unsuitable for HTS/HCS. The z-score plate-based statisticalscoring method may be used as described previously to identify compoundsthat behaved as statistical outliers compared to the other substances(n=320, no controls) tested on an assay plate for selected HCSmulti-parameter measurements output by the image analysis module (62).The z-score=(X_(i)− X)/σ, where X_(i) is the raw measurement on the ithcompound, and X and σ are the mean and standard deviation of all thesample measurements on a plate. The B-score may be calculated from allof the sample measurements on an assay plate and used an iterativemathematical model to eliminate systematic row and column artifacts on aplate. The mathematical model of the B-score may be described as:

Y _(ijp)=μ_(ijp) +YR _(ip) +YC _(jp)+ε_(ijp)

where Y_(ijp) is the compound measurement at i_(th) row and j_(th)column of the p_(th) plate, μ_(ijp) is the ‘true’ activity value,ε_(ijp) is the random error of the assay on the p_(th) plate, andYR_(ip) and YC_(jp) represent the row and column artifacts on the pthplate, respectively. A two-way median polish statistic method may beapplied to estimate the B-score of a HCS assay. The random errorestimate, ε_(ijp), of the measurement at i_(th) row and j_(th) column ofthe p_(th) plate may be calculated by fitting a two-way median polishas:

{circumflex over (ε)}_(ijp) =Y _(ijp) −Ŷ _(ijp) =Y _(ijp)−({circumflexover (μ)}+{circumflex over (R)} _(ip) +Ĉ _(jp))

where Ŷ_(ijp) is the fitted compound value, {circumflex over (μ)} is theestimated average of the plate, and {circumflex over (R)}_(ip) andĈ_(jp) are the estimated systematic artifacts for the i_(th) row onp_(th) plate and j_(th) column on p_(th) plate, respectively. Next themedian absolute deviation (MAD) of the random error estimate on p_(t),plate may be computed as:

MAD_(p)=Median{|{circumflex over (ε)}_(ijp)−Median({circumflex over(ε)}_(ijp))|}

At the last step, the B score may be calculated as:

${B - {score}} = \frac{{\hat{ɛ}}_{ijp}}{M\; A\; D_{p}}$

The compounds may be ranked according to ascending B-score values.Rank-scores may be calculated by taking the average of compound rankingsfrom two independent drug screens. Compounds with rank-scores <110 maybe considered to significantly decrease the accumulation of sGFP::ATZinclusions. Conversely, compounds with rank-scores >1225 may beconsidered to significantly increase the accumulation of sGFP::ATZinclusions. Selected compounds may be chosen for further analysis, forexample in a human cell culture and/or animal model assay.

The foregoing specific examples may readily be modified to study otherproteins with a tendency to aggregate according to the invention.

6. EXAMPLE High Throughput Genetic and Drug Screens for α1-AntitrypsinDeficiency Using C. elegans

Several characteristics of AT deficiency make it an attractive targetfor chemoprophylaxis strategies involving high-throughput screening ofsmall molecule libraries. First, the disease predominantly involves anER translocation defect, wherein mutant ATZ protein retains some of itsanti-elastase activity (i.e., ATZ can inhibit neutrophil elastase albeitnot as efficiently as AT). Thus, small molecules that increase ATZsecretion could theoretically prevent tissue damage in both lung andliver. Second, the severity of tissue injury caused by ATZ is thought tobe influenced by genetic and environmental modifiers that regulateendogenous quality control mechanisms for disposal of misfoldedproteins. Compounds that enhance these degradative processes couldtherefore be used in patients to prevent liver damage in combinationwith strategies specifically designed to prevent lung damage.

A tractable genetic model of this disease would greatly enhance theability to elucidate the mechanism of tissue damage and the endogenousmechanisms that protect against protein misfolding. As shown by theresults of experiments described below, the conformational disease of ATdeficiency can be modeled in C. elegans. Animals expressing wild-type AT(ATM) secrete the protein. In contrast, animals expressing ATZ developintracellular inclusions and show a slow growth and larvalarrest/lethality phenotype. Further, an assay using this model has beenadapted to allow for automated high-throughput screening.

Many different abbreviations have been used in the literature todesignate α1-antitrypsin (AT) and its mutant alleles. To avoidconfusion, the nomenclature for mutant and wild-tune AT alleles usedherein is described in TABLE 1,

TABLE 1 AT allele/gene product abbreviations abbreviation description ofallele and gene product AT generic for antitrypsin, does not designate aspecific allele ATM wild-type M allele, also referred to as AT in manypublications ATZ classical Z mutant with E342K mutation that ispolymerogenic and accumulates in the ER ATM-Saar 32 amino acidC-terminal truncation mutant that is non-polymerogenic and accumulatesin the ER; referred to as AT Saar in our previous publications. ATZ-Saar32 amino acid C-terminal truncation mutant that also has the pointmutation of ATZ, but is non- polymerogenic and accumulates in the ER;referred to as AT saar Z in our previous publications. ATS E264V—secondmost common mutation ATI R39C—decreased inhibitory activity, milddeficiency ATM_(malton) TTC deletion/DF52—shorter polymers in plasma@2-3 units; better secreted than Z ATM_(siiyama) S53F—polymers inplasma@15-20 units; second site mutation (F51L) enhances secretionM_(siiyama) >> Z AT_(Hong Kong) TC deletion; L318fsX334, ERAD substrate;loss 61 aa; not secreted

Construction of ATZ Expression Plasmids.

To readily visualize ATZ aggregates in live animals without the need forcomplex immunological staining methods, ATZ was fused to the greenfluorescent protein (GFP). Although, GFP-fusions have been usedextensively in cell culture and other systems, their usefulness instudying ATZ aggregation has not been tested in C. elegans. Since ATZaggregation is thought to occur via insertion of the reactive site loop(RSL) of one molecule into the n-sheet of another, it was unclearwhether GFP would interfere with this process. To assess this, N- andC-terminal GFP-ATZ fusion constructs were engineered. The nhx-2 promoter(Pnhx-2), which drives expression specifically in the intestinal cellsthrough all stages of postembryonic development, was chosen to directexpression of the fusion construct. The C. elegans intestine is thecenter of metabolic activity and the organ that most closely resemblesthe human liver (where ATM is normally synthesized). In addition, theintestine would be the predominant site of absorption for compoundsadded to the media. To direct transgene expression to the ER-Golgisecretory pathway, the synthetic signal peptide from the C. elegansexpression vector, pPD95.85, was used (Fire Lab C. elegans Vector Kit,1995).

Initial expression studies using human AT cDNA were hampered by lowexpression of the transgene. In C. elegans, transgene expression isquantitatively enhanced by the presence of intronic sequences. As such,genomic DNA is preferred over cDNA. The large gene size and variationsin RNA splicing requirements between human and C. elegans make itimpractical to use genomic ATZ DNA for expression studies in C. elegans.To improve ATZ expression, synthetic introns resembling those found inC. elegans were introduced into the ATZ cDNA. Typically, introns areintroduced by engineering unique blunt end restriction enzyme sites inthe cDNA by site-directed mutagenesis. This approach is labor intensive,inefficient and dictated by the presence of favorable sequences. As analternate approach, a strategy was devised for intron insertion usingoverlap-extension PCR (FIG. 1A-B). Large oligonucleotides consisting of˜50 nt of synthetic intron (carefully designed to contain appropriate 5′and 3′ donor/acceptor sequences) and ˜22 nt sequence complementary tothe ATZ coding region were synthesized and used as primers to amplifysmall regions of the ATZ cDNA (FIG. 1A). The amplified fragments werejoined pairwise using overlap-extension PCR to generate larger fragmentscontaining intronic regions (FIGS. 1B and 1C). Once the 5 pieces werejoined together, the complete ATZ fragment containing all of thesynthetic introns was amplified using primers flanked with Kas Irecognition sites (FIG. 1D) and cloned into the expression vectorpPD95.85 to generate Pnhx-2::sGFP::ATZ. A control construct designed toexpress ATM, Pnhx-2::sGFP::ATM, was also generated. Prior to theinsertion of the ATZ fragment, several modifications were made topPD95.85 vector to facilitate cloning and to ensure proper transgenefusion. First, a unique Kas I restriction enzyme site was introduced 3′to the GFP coding sequence to accommodate insertion of ATZ. Second, thestop codon of GFP was removed to ensure read-through to the downstreamATZ gene. All of the constructs were sequenced to confirm the absence ofmutations and in-frame insertion of coding regions. In addition to theATZ and wild-type alleles (ATM), a series of constructs were generatedcontaining several other well-described AT mutations to determine howtheir phenotypes compare to that of ATZ. A list of all the AT constructsis shown in TABLE 2. Studies comparing the position (N- or C-terminus)of the GFP fusion indicated that N-terminal GFP::AT fusions were moreefficiently expressed. As such, all subsequent AT expression constructswere generated with N-terminally fused GFP. In addition to AT, anotherserpin aggregation disorder called Familial encephalopathy withneuroserpin inclusion bodies (FENIB) has also been modeled in C.elegans. Naturally occurring mutations cause neuroserpin to aggregate ina manner similar to that of ATZ. Thus, a C. elegans model of FENIB wouldbe a valuable tool for the study of neuroserpin aggregation and theidentification of therapeutic drugs. Constructs used to generateneuroserpin transgenic worms are shown in TABLE 3.

TABLE 2 AT constructs used to generate transgenic worms Name PromoterSig GFP Description Pnhx-2::sATM intestine No none Secreted ATM (no GFP)Pnhx-2::sATZ intestine No none Secreted ATZ (no GFP) Pnhx-2::GFPintestine No — Intracellular expression of GFP Pnhx-2::GFP::ATMintestine No N-terminal Intracellular expression of GFP::ATMPnhx-2::GFP::ATZ intestine No N-terminal Intracellular expression ofGFP::ATZ Pnhx-2::sGFP intestine Yes Secreted GFP (control)Pnhx-2::sGFP::ATM intestine Yes N-terminal Secreted GFP::ATMPnhx-2::sGFP::ATZ intestine Yes N-terminal Secreted GFP::ATZPnhx-2::sATM::GFP intestine Yes C-terminal Secreted ATM::GFPPnhx-2::sATZ::GFP intestine Yes C-terminal Secreted ATZ::GFPPnhx-2::sGFP::ATM.saar intestine Yes N-terminal Secreted GFP::ATM.saarPnhx-2::sGFP::ATZ.saar intestine Yes N-terminal Secreted GFP::ATZ.saarPnhx-2::sGFP::ATS intestine Yes N-terminal Secreted GFP::ATSPnhx-2::sGFP::ATI intestine Yes N-terminal Secreted GFP::ATIPnhx-2::sGFP::ATM_(malton) intestine Yes N-terminal SecretedGFP::ATM_(malton) Pnhx-2::sGFP::ATM_(siiyama) intestine Yes N-terminalSecreted GFP::ATM_(siiyama) Pnhx-2::sGFP::AT_(Hong Kong) intestine YesN-terminal Secreted GFP::AT_(Hong Kong) Psrp-2::GFP hypoderm No —Intracellular expression of GFP (control) Psrp-2::sGFP hypoderm YesN-terminal Secreted GFP (control) Psrp-2::sGFP::ATM hypoderm YesN-terminal Secreted GFP::ATM Psrp-2::sGFP::ATZ hypoderm Yes N-terminalSecreted GFP::ATZ

TABLE 3 Neuroserpin (NS) expression constructs Name Promoter SigDescription Pnhx-2::sGFP::NS intestine Yes Wild-type neuroserpinPnhx-2::sGFP::NS(S49P) intestine Yes Polymerigenic mutantPnhx-2::sGFP::NS(G392E) intestine Yes Polymerigenic mutantPunc-119::sGFP::NS pan-neuronal Yes Wild-type neuroserpinPunc-119::sGFP::NS(S49P) pan-neuronal Yes Polymerigenic mutantPunc-119::sGFP::NS(G392E) pan-neuronal Yes Polymerigenic mutantPmec-4::sGFP::NS mechanosensory neurons Yes Wild-type neuroserpinPmec-4::sGFP::NS(S49P) mechanosensory neurons Yes Polymerigenic mutantPmec-4::sGFP::NS(G392E) mechanosensory neurons Yes Polymerigenic mutantPodr-10::sGFP::NS chemosensory neurons Yes Wild-type neuroserpinPodr-10::sGFP::NS(S49P) chemosensory neurons Yes Polymerigenic mutantPodr-10::sGFP::NS(G392E) chemosensory neurons Yes Polymerigenic mutant

Generation of ATZ Transgenic Animals.

Transgenic animals expressing AT were generated by injecting theplasmids in TABLE 2, into the gonads of young adult hermaphrodites at aconcentration of 80 ng/μl. GFP-positive progeny were selected andpropagated to confirm germline transmission. Injected DNA typicallyforms extrachromosomal arrays that are transmitted (at rates rangingfrom 0-95%) to subsequent generations in a non-Mendelian manner. Stable,integrated lines, with 100% transmission rates, can be generated byexposing animals to high doses of gamma radiation. Apart from obviatingthe need for constant selection of GFP-positive worms under thefluorescent microscope, worms with integrated arrays display more stableand consistent transgene expression. Moreover, integrated lines areparticularly advantageous for genetic screens and for experimentsrequiring analysis of large populations. For these reasons, stable,integrated lines were generated by exposing transgenic animals to 35Gy(3500 Rads) of gamma radiation. After stable integrants were identified,they were outcrossed 6 times to remove spurious mutations that may havebeen acquired as a result of the radiation treatment.

Although the intestinal specificity of the nhx-2-promoter has beendescribed, the fate of intestinally secreted GFP (sGFP) was unknown. Toaddress this issue, two constructs, Pnhx-2::GFP and Pnhx-2::sGFP, wereprepared as positive controls for GFP secretion. Transgenic animalsharboring Pnhx-2::GFP plasmids showed strong GFP expression in theintestinal cells (FIG. 2A). Transgenic animals harboring Pnhx-2::sGFPshowed no visible GFP expression in the intestinal cells (FIG. 2B).Instead, diffuse GFP expression was seen in the pseudocoelomic space(FIG. 2B, asterisks). A similar expression pattern was observed inanimals expressing ATM (Pnhx-2::sGFP::ATM) (FIG. 2C). These dataindicated that the synthetic signal peptide of pPD95.85 was sufficientfor directing the transgene to the ER-Golgi secretory pathway. Incontrast, animals harboring the ATZ transgene (Pnhx-2::sGFP::ATZ) showednon-detectable levels of sGFP::ATZ in the pseudocoelomic space. Instead,bright intracellular GFP positive inclusions were observed (FIGS. 2D and2E, red arrows), suggesting intracellular retention and aggregation.These observations closely resemble previous findings in human cellculture and mouse models and indicated that ATZ aggregation can berecapitulated in C. elegans.

To determine if correct fusion proteins were being synthesized, totalworm lysates were analyzed by immunoblotting. Mixed staged worms werelysed by sonication. Following centrifugation to remove cell debris,worm lysates were boiled in sample buffer for 5 min and fractionated bySDS-PAGE. Gels were transferred to nitrocellulose membranes and AT andGFP protein bands were visualized by probing with anti-AT and anti-GFPantibodies, respectively (FIG. 3). The anti-human AT antibody did notreact with any proteins in the lysates of the parental N2 (lane 1) orcontrol sGFP (lane 2) expressing worms (FIG. 3A). In contrast, a proteinband migrating at ˜50 kDa, corresponding to the purified plasma AT, wasrecognized (lane 5). In addition, a protein band migrating at ˜80 kDawas detected in the lysates of worms expressing sGFP::ATM (lane 3) andsGFP::ATZ (lane 4) (FIG. 3A). To confirm that the ˜80 kDa protein bandscorresponded to sGFP::ATM and sGFP::ATZ fusion proteins, a parallel blotwas probed with an anti-GFP antibody (FIG. 3B). Identically-sizedprotein bands were recognized by both antibodies (lanes 3 and 4)indicating that the ˜80 kDa protein bands were indeed sGFP::ATZ or ::M)fusions.

To further characterize the nature and location of the ATZ globules,worms were fixed in glutaraldehyde, stained with osmium tetroxide andexamined under an electron microscope (FIG. 4). No abnormal structureswere observed in the intestinal cells of worms expressing sGFP::ATM(FIG. 4A). In contrast, large intracellular globules were present insections of worms expressing sGFP::ATZ (FIGS. 4B and C, arrowheads).These globules appeared to be surrounded by ribosomes (FIG. 4D),suggesting retention of the ATZ proteins within the ER.

Development of a fully automated, high-content, high-throughput, wholeorganism-based screen for drugs and RNAis. C. elegans is a powerfulgenetic organism useful for the study of human diseases. Recently, therehave been growing interest in using C. elegans as a tool in drugdiscovery. Efforts have been hampered by one major bottleneck—automatedimage capture and data analysis. The present invention now provides afully automated, whole organism-based high-content screen (HCS) fordrugs and RNAis modulating the disease phenotype. This method can beeasily adapted to other C. elegans models and provides a stepping-stonefor future C. elegans-based drug and RNAi screens.

The ability to conduct a high throughput drug screen depends largely onthe capacity to rapidly and accurately detect and quantify the givenphenotype. Prior to the present invention, automated image capturetechnology has focused largely on cells in culture. Image capture ofmulticellular, whole organisms is presented with numerous challengessuch as autofocusing of the region of interest.

To determine whether the C. elegans model of ATZ could be used forhigh-content, high-throughput screens, a 96 well-based assay wasdeveloped. Fifty-five young adult stage worms were dispensed into96-well plates using the COPAS BioSort (worm sorter). Worms were thenimmobilized by the addition of 0.1 M sodium azide to facilitate imagecapture. The plates were placed into a computerized high throughputplate reader, ArrayScanVTi (Thermofisher Cellomics Products).ArrayScanVTi was set up to rapidly scan wells and capture multipleimages using bright field and fluorescence parameters. Complexalgorithms were set up to identify and quantify defined objects(fluorescent granules) and events (individual animals). A typical screenshot of the ArrayScanVTi interface is shown in FIG. 5. In this case,bright field and GFP fluorescence images were taken from 4 differentfields within each well using a 5× Carl Zeiss objective. A single fieldshowing a bright field and GFP fluorescence overlay illustrates GFPaggregates throughout the length of the intestine (FIG. 5, well D1). Toquantify these GFP aggregates, algorithms were developed to firstidentify the objects of interest (adult worms) and quantify the numberand intensity of the spots (aggregates). FIG. 6 shows exactly theelements from FIG. 5 that were selected for data analysis. ArrayScanVTicorrectly identified all adult worms in the field of view (blue outline)and excluded all eggs and other debris that would alter the analysis.Moreover, the algorithm identified all the ATZ aggregates (FIG. 6, redspots) within the set boundary (FIG. 6, red outline).

After defining the parameters to accurately identify worms andaggregates within ATZ worms, the sensitivity of the approach todiscriminate between N2, sGFP::ATM and sGFP::ATZ animals was tested. Inall cases, the algorithm identified correctly all adult worms in thefield of view (FIG. 7, blue outline). As expected, no GFP aggregateswere identified in N2 animals and a small number of “aggregates” wereidentified in sGFP::ATM animals. In contrast, numerous aggregates wereidentified within ATZ animals (FIG. 7, ATZ, red spots). To quantify thedifferences between the types of animals, the total number of spots andthe total and average spot area for all 4 fields were determined by thealgorithm (FIG. 8). Consistent with the fluorescence images, noaggregates (spots) were identified in the N2 animals. In contrast, >450aggregates were identified in the well containing the sGFP::ATZ animals.A small number (<70) of spots were identified in the sGFP::ATM animals,however, the total and average spot areas were <5% and <20% that of ATZanimals, respectively. These results indicated that ATZ aggregates canbe readily identified and quantified. These data also demonstrated thesensitivity of the system in detecting smaller and less intenseaggregates, parameters that may be important for identifying compoundsthat induce a partial reduction in ATZ aggregation.

Although the above-mentioned method could discriminate between thevarious transgenic lines, the initial assay using the brightfield modulewas plagued with long focus times, inconsistent object identificationand inaccurate determination of worm number per well. These problems arenot limited to the Arrayscan VTi but to brightfield imaging in general.To circumvent these problems, a new transgenic AT lines that expresses ared fluorescent protein (mCherry) in the head (pharynx) region of theworm was developed (FIG. 9). The expression of a second fluorescentmarker that has a distinct expression pattern than GFP::ATZ (intestine)has several major advantages. First, the bright expression of themCherry protein significantly improves focus time and efficiency.Second, with optimized algorithms, red heads can be easily counted toobtain accurate worm number per well. Thirdly, since the pharynx is inthe same focal plane as the intestine, GFP::ATZ aggregates can be moreefficiently and accurately measured. By simply dividing the total GFPfluorescence in the well by the total number red heads, the average GFPfluorescence per worm can be determined, a capability that wasproblematic to achieve using the brightfield object identificationalgorithms.

Initial feasibility experiments were performed using 96-well plates. Toincrease the high-throughput capacity of the assays, a 384-well-basedassay was developed. A 384-well-based assay has several advantages overthe 96-well format. First, one can screen more compounds using the samenumber of worms needed for a 96-well plate. Second, images can becaptured using the 2.5× objective. This reduces the number of fieldsneeded to capture the well from 16 to 1. In addition, the Arrayscan VTiwas fitted with a 0.63× coupler. The 0.63× coupler allows the capture of100% of the well (as opposed to ˜90% using the 1× coupler) allowing oneto account for all the worms on in the well. This has lead to aconsiderably smaller variance between replicate wells.

Collectively, the ability to utilize an automated detection system tomeasure changes in the size and number of ATZ aggregates in individualanimals cultured in 384-well plates removes the single most significantbottleneck/obstruction to the development of high throughput screeningusing a whole animal such as C. elegans.

A Small Molecule Screen of the LOPAC Library Identifies DrugsPotentially Useful for the Treatment of AT-Deficiency and Other ProteinAggregation Disorders.

To determine whether the C. elegans model could be used in ahigh-content, high-throughput drug screen context for modifiers ofmisfolded ATZ accumulation, a pilot screen of the LOPAC (library ofpharmacologically active compounds) library was performed. The globalstrategy for high-throughput screen is shown in FIG. 10A-E.

Two μl of stock (10 mM) compounds were diluted with 98 μl of S-medium toa final concentration of 200 μM drug in 2% DMSO and S-medium. Fifteen μlof the diluted compounds were then transferred to 384-well plates usinga robotic liquid handler (EP3). Prior to the experiment, fifteen μl of4×OP50/antibiotic solution were added to each well. Using the COPASBiosort, 35 L4-young adult stage worms were deposited into each well andallowed to incubate for 24 or 48 h at 22° C. At the end of theincubation period, worms were immobilized by the addition of sodiumazide or levamisole to a final concentration of 12.5 mM and 4 mM,respectively. The worms were then imaged using the high speed, automatedimaging device, ArrayScan VTi. Image capture and data analysis wereperformed using the Spot Detector BioApplication with algorithmsoptimized for worms. B-score statistical analyses were performed toidentify compounds that had a significant (>2 SDs away from the mean)effect on ATZ aggregation. A summary of a typical LOPAC screen is shownin FIG. 11. Using the above approach, three screens of the LOPAC librarywere performed which identified a number of hit compounds that havepotential therapeutic value (TABLE 4).

TABLE 4 List of hit compounds Mol ID Name Description 75 W-7hydrochloride Calmodulin antagonist 140 H-89 cAMP-dependent proteinkinase (PKA) inhibitor 248 Cantharidin Protein phosphatase 2A inhibitor249 Chlorpromazine Dopamine receptor antagonist; hydrochlorideanti-emetic; antipsychotic 275 4-Chloromercuribenzoic Carboxy- andaminopeptidase acid inhibitor 280 Pyrocatechol Carcinogen; causes DNAstrand breakage 291 CGP-74514A hydrochloride Cdk1 inhibitor 318Cantharidic Acid Protein phosphatase 1 (PP1) and 2A (PP2A) inhibitor 396Dequalinium analog, Protein kinase C-alpha C-14 linker (PKC-alpha)inhibitor 553 (−)-Eseroline fumarate Metabolite of physostigmine(eserine); potent analgesic; cholinesterase inhibitor 555 FluphenazineDopamine receptor antagonist; dihydrochloride antipsychotic 600Idarubicin Antineoplastic 799 Se-(methyl)selenocysteine Potentchemopreventive agent hydrochloride 946 Pimozide Ca2+ channelantagonist; antipsychotic; D2 dopamine receptor antagonist * listed inorder of Mol ID

7. EXAMPLE Automated High-Content Live Animal Drug Screening Using C.elegans 7.1 Materials and Methods

Construction of Promoter-Transgene Fusions:

A transcriptional Pmyo-2mRFP fusion construct was constructed bysubcloning the myo-2 promoter and the mRFP cDNA into the SphI/XbaI andNheI/EcoRV sites of the canonical expression vector, pPD49.26 (a kindgift from Dr. Andrew Fire, Stanford University School of Medicine),respectively. To generate the Pnhx-2mCherry::lgg-1 construct, a 3.5 kbgenomic fragment containing the lgg-1 promoter, coding region and 3′-UTRwas amplified and cloned into pCR®-Blunt II-TOPO® vector (Invitrogen,Carlsbad, Calif., USA). Using site directed mutagenesis a unique MluIrestriction enzyme site, was introduced upstream of the lgg-1translation start codon. The mCherry cDNA, lacking a translation stopcodon, was inserted into the MluI site, which places it in-frame withthe lgg-1 coding region. To direct expression of the mCherry::lgg-1fusion gene in intestinal cells, we replaced the lgg-1 promoter with a1.5 kb nhx-2 promoter using a HindIII restriction site. Pnhx-2sGFP::ATMwas generated by inserting a 4 kb nhx-2 promoter fragment intoHindIII/XbaI restriction sites of the expression vector, pPD95.85. Thena KasI restriction site was introduced by site-directed mutagenesis intothe GFP translational stop codon. A 1.4 kb fragment containing the ATMcDNA and 3 synthetic introns was then cloned into the KasI site.Pnhx-2sGFP::ATZ was generated by site-directed mutagenesis ofPnhx-2sGFP::ATM, thereby generating the E342K (Z) mutation. The plasmidcontaining Pnhx-2GFP, pFH6IInhx-2, was a kind gift from Keith Nehrke(University of Rochester Medical Center) (95).

Worm Strain and Culture Conditions:

Worm strains: VK413 (Pnhx-2GFP), VK1093 (Pnhx-2mCherry::lgg-1), VK821(Pmyo-2mRFP) were generated by injecting the respective plasmids intothe gonad of young adult N2 hermaphrodites at a final concentration 80ng/μl. Strains VK689 (Pnhx-2sGFP::ATM) and VK694 (Pnhx-2sGFP::ATZ) weregenerated by co-injecting the plasmids and Pmyo-2mRFP at a finalconcentration of 70 ng/ml and 10 ng/ml, respectively. The worm strainexpressing Pclh-4GFP (pFL6IIclh-4) were a gift from Keith Nehrke 40. N2and GF66 (Pvha-4Q82::YFP, 21) were obtained from Caenorhabditis GeneticsCenter (CGC), http://www.cbs.umedu/CGC/). Worms were routinely culturedat 22° C. on nematode growth medium (NGM) plates seeded with E. colistrain, OP50, unless otherwise specified.

Imaging of Transgenic Animals Using ArrayScan VTI:

Twenty N2 or transgenic L4-adult stage worms were transferred to384-well plates containing 60 μl of PBS and anesthetized with 30 μl of0.02 M NaAz prior to image capture. Images were acquired with theArrayScan VTI HCS Reader (Cellomics, ThermoFisher, Pittsburgh, Pa., USA)fitted with a 5× or 2.5× objective and a 0.63× coupler. For thedetection of various developmental stages using N2 worms, images werecaptured using the brightfield channel. Valid objects (adult worms) wereautomatically selected using the SpotDetector BioApplication(Cellomics). For image capture and analysis of the lines expressingfluorescent transgenes, we employed a 2-channel (brightfield and GFP orTRITC) assay. Algorithms were optimized to first identify valid objects(blue outline in FIGURES.), defined as non-overlapping, whole worms inthe brightfield channel. Debris and partial worms were automaticallyexcluded (orange outline in Figs.) from analysis. Fluorescent transgeneexpression, within valid objects, was quantified in the TRITC or GFPchannels. SpotDetector BioApplication was optimized to identifytransgene expression as spots. Parameters were optimized such that spotsof varying shape, size and intensity could be identified. For thispaper, spot count, spot total area and spot total intensity per objectwere used to compare transgene expression in different animals.

Whole Animal Alive-Dead Assay:

Adult N2;Is1033[Pmyo-2::mRFP] animals were incubated at room temperaturewith sodium azide (0-100 mM) for 4 hours. Animals were washed 5 timeswith M9 media and stained with 2 μM SYTOX™ Green (Invitrogen) for 5minutes at room temperature (84). Approximately 50 animals/well weredispensed into an optical bottom black walled 96 well plate (Nunc ThermoFisher Scientific, Rochester, N.Y., USA). Wells were imaged using theArrayScan VTI over the entire area of the well in brightfield, red(TRITC) and green (GFP) channels at 50× magnification. The total numberof animals and the number of dead bodies were determined by counting redand green spots, respectively. Data from SpotDetector algorithm wereconfirmed by manual counting of the live and dead animals. Percentdead=(the number of green objects detected/total number of animals)×100.

OP50 Preparation for Growth of Animals in Liquid Culture:

A single colony of OP50 was placed in 3 ml LB broth and incubated at 37°C. with vigorous shaking overnight. One milliliter of overnight culturewas added to 1 L sterile LB broth and was incubated at 37° C. withvigorous shaking until reaching an OD600=0.5. The bacteria were washedtwice with PBS and concentrated to an OD600=10.0. An equal volume of 50%glycerol was added for long-term storage at −80° C. After thawing, thebacteria were concentrated by centrifugation and re-suspended in PBS toan OD600=10.0.

Preparation of Animals for HCS Drug Screening:

Ten adult animals were placed on twelve 10 cm plates of NGM agar mediaspread with a lawn of E. coli strain OP50 (NGM/OP50). Approximately 7days later, young adult stage F2 animals were isolated by differentialsedimentation and transferred to 12 NGM/OP50 plates. After an overnightincubation at 22° C., adults were washed off with PBS and the remainingeggs were allowed to hatch overnight. Early-stage larvae weretransferred to 48 NGM/OP50 plates and allowed to grow until most of theworms were in the 4th larval (L4) stage. Using the COPAS™ BIOSORT (UnionBiometrica, Holliston, Mass., USA) approximately 15,000 L4 stage animalsexpressing similar levels of GFP were sorted into twelve 10 cm NGM/OP50plates. After an overnight incubation at 22° C., gravid adults werewashed off and transferred to fresh NGM/OP50 plates and allowed to layeggs for 5 hours. Following this incubation period, adults were washedoff and discarded leaving a synchronous population of eggs on theplates. The eggs were incubated at 22° C. for 40 hours or until themajority of the worms were in the L4/young adult stages. This methodgenerated a population of ˜200,000 age-synchronized animals for smallmolecule screening.

In preparation for sorting, animals were washed off plates andtransferred into 50 ml conical tubes and allowed to settle by gravityfor 5 minutes. After discarding the supernatant, animals were washedagain with 50 ml of PBS to remove excess bacteria and other debris thatcould interfere with worm sorting. Following the final rinse, total wormcount was determined by taking aliquots of the worm suspension. Thefinal worm concentration was routinely adjusted to ˜400 animals/ml.

Compound Libraries and Handling, Dilution and Transfer to Assay Plates:

The 1280 compound Library of Pharmacologically Active Compounds (LOPAC)was purchased from Sigma-Aldrich (St. Louis, Mo., USA). Compounds werearrayed into 384-well microtiter master plates at a concentration of 10mM in DMSO. LOPAC compounds were given unique University of PittsburghDrug Discovery Institute (UPDDI) substance identity numbers and werehandled and stored as described (95-99). Daughter plates containing 2 μlof 10 mM compounds in DMSO were prepared and replicated from the LOPACmaster plates using the Vprep (Agilent Technologies, Santa Clara Calif.,USA) outfitted with a 384-well transfer head. Aluminum adhesive plateseals were applied with an Abgene Seal-IT 100 (Rochester, N.Y., USA)plate sealer and plates were stored at −20° C. in a MatricalMatriMinistore™ (Spokane, Wash., USA) automated compound storage andretrieval system. For the primary screen, daughter plates were withdrawnfrom the −20° C. freezer, thawed at ambient temperature and centrifuged1-2 min at 50×g. The plate seals were removed and 98 μl of S-medium wereadded to the wells using the Flex Drop dispenser (Perkin Elmer, Waltham,Mass., USA). This intermediate stock of library compounds was at aconcentration of 200 μM in 2% DMSO. The diluted compounds were mixed byrepeated aspiration and dispensation using a 384-well P30 dispensinghead on the Evolution-P3 (EP3) liquid handling platform (Perkin Elmer),and then 15 μl of each compound were transferred to the wells of assayplates. In the primary screen, compounds were screened individually at afinal concentration of 50 μM.

Assay Plate Preparation for Drug Screen:

On the day of the screen, assay plates containing 15 μl of each compoundwere thawed and centrifuged at 214×g for 60 s. Fifteen microliters of 4×assay medium, which was prepared by mixing 4.0 ml OP50, 25.4 mlS-medium, 0.6 ml 100× antibiotic-antimycotic stock solution (stockcontained 10,000 units penicillin, 10 mg streptomycin and 25 μgamphotericin B/ml, Sigma) and 24.0 μl 1 M FUDR, were added to each well.Animals were then sorted into the wells using the COPAS™ BIOSORT wormsorter.

Animal Sorting Using the COPAS™ BIOSORT:

To reduce assay variability, a tightly-synchronized population of wormswas selected based on size (i.e., stage of development) and fluorescenceintensity (i.e., transgene expression) using the COPAS™ BIOSORT. L4 toyoung adult-stage worms were initially selected usingempirically-determined time-of-flight (TOF) and coefficient ofextinction (EXT) values. Animals were also gated based on GFPfluorescence intensity. Approximately 30% of the starting population wasselected.

For analytical assays, animals were suspended in S-medium (minus EDTA)for sorting. The flow rate was maintained at ˜25 worms/sec. Coincidencecheck was employed to enhance selection specificity. For LOPAC libraryscreening, COPAS sheath fluid was replaced with 0.01% Triton X-100 inS-medium (minus EDTA) to promote healthy bacteria and worm growth.Thirty-five L4 to young-adult animals were sorted into wells containingcompounds and assay medium. The final total volume per well afteraddition of the animals was 60 Approximately 45,000 worms were requiredfor each 384-well plate. On average, sorting time was 90 minutes perplate. The flow cell was periodically flushed between plates to preventclogging. Four 384-well plates were routinely sorted on the same day.Plates were then sealed with ThinSeal T-2417-4 (ISC BioExpress,Kaysville, Utah, USA) and incubated at 22° C. for 24-48 hours.

Imaging of Animals Using the ArrayScan VTI:

Prior to imaging, worms were anesthetized by adding 30 μl of 0.02 M NaN3in PBS to each well. Plates were resealed, inverted twice, and incubatedfor 5 minutes at room temperature. Images were acquired with theArrayScan VTI HCS Reader fitted with a 2.5× objective and a 0.63×coupler using a 2-channel (TRITC and GFP) assay. Real-time analysis wasperformed using the SpotDetector BioApplication optimized to quantifyfluorescent protein expression in C. elegans. Image acquisition andanalysis of a 384-well plate was completed in <45 minutes. The totalnumber of animals in the well was determined by counting the number ofred heads (Pmyo-2mRFP) in the TRITC channel. Total spot area or totalspot intensity was determined by quantifying the GFP-positive spots inthe GFP channel. Total spot area or total spot intensity per animal wasdetermined by dividing the values from the GFP channel by that from theTRITC channel.

HCS Data Analysis:

Compound tracking and data analysis for the primary HCS assay wereperformed using ActivityBase™ (IDBS, Guildford, UK), CytoMiner (UPDDI)software and visualized using Spotfire™ DecisionSite® (TIBCO SoftwareInc., Somerville, Mass., USA) software, as described in 60-63. Customcalculators were written to process the HCS data and perform the z-scoreand B-score statistical analysis (100, 101).

As a measure of assay quality and robustness, the Z′-factor 30 was used.The Z′-factor was calculated from the mean and the standard deviation ofthe negative and positive control populations as follows:

Z′=1−((3×(σ_(p)+σ_(n)))/(∥_(p)−μ_(n)))

where σ is the standard deviation, μ is the mean and p and n arepositive and negative controls, respectively. Z′-factors between 0.5 and1.0 indicate the separation band (signal window) between the positiveand negative controls is wide and the assay is of excellent quality andsuitable for HTS/HCS. Z′-factors between 0 and 0.5 indicate a goodquality screen, whereas a score <0 indicates the assay is of poorquality and unsuitable for HTS/HCS.

The z-score plate-based statistical scoring method was used as describedpreviously to identify compounds that behaved as statistical outlierscompared to the other substances (n=320, no controls) tested on an assayplate for selected HCS multi-parameter measurements output by the imageanalysis module (98). The z-score=(X_(i)− X)/σ, where X_(i) was the rawmeasurement on the ith compound, and X and a were the mean and standarddeviation of all the sample measurements on a plate.

The B-score was calculated from all of the sample measurements on anassay plate and used an iterative mathematical model to eliminatesystematic row and column artifacts on a plate. The mathematical modelof the B-score was described as:

Y _(ijp)=μ_(ijp) +YR _(ip) +YC _(jp)+ε_(ijp)

where Y_(ijp) was the compound measurement at i_(th) row and j_(th)column of the p_(th) plate, μ_(ijp) was the ‘true’ activity value,ε_(ijp) was the random error of the assay on the p_(th) plate, andYR_(ip) and YC_(jp) represented the row and column artifacts on the pthplate, respectively. A two-way median polish statistic method wasapplied to estimate the B-score of a HCS assay. The implementedprocedures are described below. The random error estimate, {circumflexover (ε)}_(ijp) of the measurement at i_(th) row and j_(th) column ofthe p_(th) plate was calculated by fitting a two-way median polish as:

{circumflex over (ε)}_(ijp) =Y _(ijp) −Ŷ _(ijp) =Y _(ijp)−({circumflexover (μ)}+{circumflex over (R)} _(ip) +Ĉ _(jp))

where Ŷ_(ijp) was the fitted compound value, μ was the estimated averageof the plate, and {circumflex over (R)}_(ip) and Ĉ_(jp) were theestimated systematic artifacts for the i_(th) row on p_(th) plate andj_(th) column on p_(th) plate, respectively. Next the median absolutedeviation (MAD) of the random error estimate on p_(th) plate wascomputed as:

MAD_(p)=Median{|{circumflex over (ε)}_(ijp)−Median({circumflex over(ε)}_(ijp))|}

At the last step, the B score was calculated as:

${B - {score}} = \frac{{\hat{ɛ}}_{ijp}}{M\; A\; D_{p}}$

The compounds were ranked according to ascending B-score values.Rank-scores were calculated by taking the average of compound rankingsfrom two independent drug screens. Compounds with rank-scores <110significantly decreased the accumulation of sGFP::ATZ inclusions.Conversely, compounds with rank-scores >1225 significantly increased theaccumulation of sGFP::ATZ inclusions. Selected compounds (based on costand availability) from both groups were chosen for further analysis.

Hit Compound Characterization:

Compounds that were identified as potential hits were purchased (ifavailable) and retested for verification. Compounds that failed toproduce a dose-dependent response were not analyzed further. Compoundsthat produced a response in a dose-dependent manner were further testedfor a time-dependent response. Compound dose-response curves wereperformed by dispensing 15 μl of a 4× stock solution into 384-wellplates containing 15 μl of assay medium (see above). Thirty-five animalswere sorted into each well bringing the volume to ˜60 μl. The finalcompound concentrations in each well varied from 0-100 μM. Assay plateswere incubated in a 22° C. incubator for 24 or 48 hours. Each compoundwas tested in quadruplicate in at least 2 independent experiments.

Statistical Evaluation:

Statistical evaluation of data was performed using Prism® (GraphpadSoftware). The significance of actual and predicted data in FIGS. 12, 14and 15 was determined using a linear regression analysis and comparingthe slope and goodness-of-fit (r2) values. Statistical significance ofthe spot count, spot area and spot intensity values between N2(wild-type) and various transgenic lines in FIG. 13 and dose-response inFIG. 17 was determined using an unpaired, one-tailed, Student's t-test.

7.2. Results

Detection of C. elegans Developmental Stages Based on Size:

An automated fluorescence microscopy imaging system by Cellomics, Inc.,originally designed for HCS and data analysis using cells(http://www.cellomics.com/content/menu/ArrayScan/), was adapted toautomate the detection and analysis of C. elegans in a 96- or 384-wellmicrotiter format. The instrument, ArrayScan VTI, consists of aninverted light microscope (Axiovert 200M, Carl Zeiss) configured with amotorized objective turret with Plan-Neofluar objectives, a motorized5-position filter cube turret, a mechanized stage, a 12-bit cooled CCDcamera and controller software. Samples are illuminated for brightfieldimaging using a broad white-light source and for florescence imaging inup to 4 different spectra using a mercury-based light source. Differenttypes of analysis modules (Thermo Scientific BioApplications)automatically convert 16-bit monochromatic images into numeric data. Todetermine whether the ArrayScan VTI and the BioApplications softwarecould distinguish accurately small animals instead of cells, the numbersof young adult C. elegans sorted into a 384-well plate were firstassessed (FIG. 12A). The software application required that objectsfirst be defined and counted in channel 1. Using brightfieldillumination, the SpotDetector BioApplication, which was programmed todetect objects of a certain size, identified nearly all the adultanimals (FIG. 12B, outlined in blue). Since the algorithm also excludesobjects of a certain size, it was determined whether, the system coulddistinguish young adult animals from eggs and the smaller L1 through L4larval forms. Populations of 36 animals, each containing differentpercentages of adult worms were sorted into 384-well microtiter plate(FIG. 12C). The SpotDetector BioApplication correctly selected (outlinedin blue) and excluded (outlined in yellow) objects of the pre-selectedsize parameters (FIG. 12D). However, some animals were not counted inwells containing a higher proportion of adults. Miscounting, whichdecreased the overall goodness-of-fit of linear regression, was due tothe inability of algorithm to resolve overlapping patterns into morethan one discrete object (FIG. 12E). As expected, the accuracy ofdetection improved when <10 adults were added to a well. Of note, theprogram can be configured to detect animals at, for example, the L1-L2stage and exclude those at the L3-L4 adult stages. Taken together, thesestudies suggested that the instrument could be used to screen forcompounds that alter the growth and development of synchronized culturesby counting the proportion of animals of a particular size at a constanttime point.

Detection of Tissues, Pathologic Subcellular Protein Aggregates andAutophagy within C. elegans:

Once valid objects are selected using the brightfield images in channel1, the ArrayScan VTI can detect fluorescent “spots” in up to 4 differentchannels within each object and the SpotDetector BioApplication candisplay the data as a total fluorescent spot number, spot area or spotintensity per object. It was next determined whether this applicationwas sensitive enough to identify different cell types (pharyngeal cells,excretory cell, intestinal cells), pathologic protein deposition (polyQaggregates) or a physiological process (autophagy) within individualobjects (animals). Fluorescent images (channel 2) were obtained for C.elegans strains carrying transgenes with tissue-specific promotersdriving fluorescent protein expression in the pharynx (Pmyo-2RFP), theexcretory gland cell (Pclh-4GFP) or intestinal cells (Pvha-6Q82::YFP,Pnhx-2GFP or Pnhx-2mCherry::lgg-1). Except for the polyQ82-containingconstruct, which generates cytosolic aggregates (78), the others yieldeda diffuse cytoplasmic fluorescence pattern under baseline conditions(FIGS. 13A-13J). In comparison to the minimal background fluorescence ofwild-type (N2) animals, that of the transgenic animals was markedlyincreased using the SpotDetector BioApplication to measure either thetotal spot number, area or fluorescence intensity per animal (FIGS.13O-13Q). Depending on the nature of the transgene expression pattern,certain comparisons were more meaningful. For example, total spot areaor total spot intensity per animal, rather than total spot count, werebetter at discriminating pharyngeal or intestinal expression incomparison to background (FIGS. 13C-13F, 13P, 13Q). In contrast, totalspot count per animal, was the more sensitive parameter to follow whenassessing the presence of the secretory cell and the degree of proteinaggregation in the animals expressing polyQ82 (FIGS. 13G-13J, 13O).

Macroautophagy is a cellular process in which a double membrane envelopscytosolic components or organelles (autophagosome) and delivers thismaterial to a lysosome (autophagolysosome) for degradation and recycling(reviewed in 79). LGG-1/LC3/Atg8 is a diffuse cytosolic protein thatparticipates in autophagosome formation and becomes inserted into theautophagosome membrane (80). Upon autophagosome formation, LGG-1 fusedto mCherry changes its cytoplasmic distribution pattern from diffuse(lower fluorescence intensity) to punctate (higher fluorescenceintensity) (80). To determine whether the imaging system could followthis process, a strain expressing a Pnhx-2mCherry::lgg-1 transgene wasexamined after starvation, a potent inducer of intestinal autophagosomeformation (81). In well-fed animals, the diffuse cytoplasmicfluorescence in the intestinal cells was well above that of the N2background (FIGS. 13K-13L, 13O-13Q). To detect mCherry::LGG-1 puncta,the diffuse fluorescence intensity of the well-fed animals was used tocalibrate a threshold from which the SpotDetector BioApplication coulddetect any high-intensity spots. Although basal autophagy in thewell-fed animals yielded a few spots (FIG. 13O), the large number ofdistinct puncta in the starved animals (FIGS. 13M, 13N, 13O-Q) indicateda marked increase in autophagy that was detected best by a statisticallysignificant increase in spot count or total spot intensity per animal(FIGS. 13O and 13Q, respectively). Taken together, this versatileimaging platform quantitatively measured several different types offluorescence patterns, thereby allowing for the interrogation of a widerrange of biological processes, such as tissue organization,proteotoxicity and metabolic functions.

Detection of Live Cells and Dead Animals.

The nematode has served as an informative system to study the geneticsof different modes of cell death. It was determined whether this imagingsystem could distinguish between live or dead cells using either theloss or gain of a fluorescent marker, respectively. mec-4, a member ofthe DEG/ENaC membrane cation channel superfamily, is expressedexclusively in the 6 mechanosensory neurons of C. elegans (82). Areporter strain containing an integrated transgene, ZB164bzIs8[Pmec-4GFP]; mec-4(+), diving GFP expression in the mechanosensoryneurons exhibits ˜4-5 fluorescent cell bodies per L4/young adult animal(83). In contrast, post-developmental necrotic cell death graduallyoccurs in most of the mechanosensory neurons after the reporter strainis crossed with animals containing a toxic gain-of-function mutation,mec-4(d). To determine whether the imaging system could distinguish thewild-type from the mec-4(d) strain, adult animals were identified bybrightfield illumination in channel 1 (FIGS. 14A, 14D, 14G), and forcomparison, by fluorescence imaging to display the GFP-labeledmechanosensory neurons in channel 2 (FIGS. 14B. 14E. 14H). SpotDetectorquantified the number of live florescent cells (spots) present in eachbrightfield object (FIGS. 14C, 14F, 14I). Consistent with previousstudies, the mec-4(+) and mec-4(d) strains averaged ˜6 and ˜2cells/animal, respectively (FIG. 14M) (45). Remarkably, the system wascapable of discriminating between wild-type and mutant animals based onthe differential viability of just six mechanosensory neurons.

Animals exposed to toxic doses of sodium azide (NaAz) undergo massivenecrotic intestinal cell death characterized by a marked loss ofmembrane permeability (84). Thus, the uptake of the membrane impermeantfluorescent nucleic acid dye, SYTOX® Green, serves as a dead cellindicator. To determine whether the system could discriminate dead fromlive intestinal cells, we scanned and analyzed young adult animalsexposed to different concentrations of NaAz in the presence SYTOX®Green. Dead animals showed extensive uptake of SYTOX® Green that wasaccurately detected by the imaging system (FIGS. 14J-14L). Analysisshowed a dose dependent increase in the number of dead animals and anexcellent correlation with the number counted manually (FIG. 14N). Itwas concluded that this automated system was capable of detecting deadcells and should prove useful in developing HCS for drugs that modulatenecrotic cell death.

Development of a HCS Protocol Using C. elegans:

Although brightfield imaging in channel I accurately detected adultanimals (objects) in the well of a 384-well plate (FIG. 12), the timerequired to autofocus and capture each animal, plus a need to limit theadult worm population to ˜10 animals per well (due to overlapping)decreased throughput and assay robustness. To obviate these problems,Pmyo-2mRFP transgenic animals that expressed the fluorescent protein intheir pharyngeal region were used (FIG. 13F). Since the totalfluorescence area or total fluorescence intensity of this region wasproportional to the overall size and developmental stage of the animals,it was determined whether fluorescence imaging of the “red-heads” usingthese parameters could be substituted for the more time-consumingbrightfield imaging. As above (FIG. 12), populations of 36 transgenicanimals were sorted, each containing different percentages of adultworms, into the wells of 384-well microliter plate. A compositebrightfield and fluorescence image showed that all of the animals had adetectable red-head that was proportional in area to the developmentalstage and size of the animal (FIG. 15A). Next, brightfield optics werepreset in channel 1 to detect the entire well as a single “object”. Oncean object was defined in channel I, the SpotDetector BioApplication wasprogrammed to select (pseudocolored red heads) or exclude (pseudocoloredwhite heads) fluorescent spots above or below, respectively, apre-determined threshold value based on a combination of fluorescentspot area and intensity (FIG. 15B). In this example, the algorithmcorrectly identified all 9 young adult animals and excluded ˜24 of thelarval forms (FIG. 15B). Since, the area of the red-heads wasproportionally smaller than that of adult animals, the total count wasrarely confounded by overlapping pharyngeal “spots”. Thus, there wasexcellent correlation between the number of adult animals detected bythe spot count and the actual number of animals in the wells (FIG. 4C).It was concluded that the number of adult animals accurately detected ina well of a 384 well plate increased from ˜10 to at least 35, whenfluorescence imaging of the red-head marker, rather than the brightfieldimaging of individual animals was used to obtain a valid animal count.

Since the imaging system can detect fluorescent “spots” in more than onechannel, it was next determined whether a combination of two differentfluorescent markers could be used to develop a high-content drugscreening strategy using live animals. First, we developed an integratedtransgenic line expressing the Z-mutation of the human secreted serpin,α1-anitrypsin (ATZ). This transgene, Pnhx2sGFP::ATZ, contains a humanATZ minigene fused C-terminal to GFP with N-terminal signal peptide(sGFP). An intestinal-specific promoter, nhx-2, drove fusion geneexpression (85). In humans, this common Z mutation induces proteinmisfolding and accumulation within the endoplasmic reticulum ofhepatocytes resulting in cellular injury and cirrhosis (reviewed in 86).Similarly, sGFP::ATZ aggregated within the endoplasmic reticulum ofintestinal cells. As a control, an integrated transgenic line,expressing the wild-type fusion protein, sGFP::ATM. was generated. Thisprotein was efficiently secreted into the intestinal lumen andpseudocoelomic space and was detectable microscopically only after arelatively long integration time. To facilitate analysis using theArrayScan VTI, both strains were co-injected with the Pmyo-2mCherrytransgene. Approximately 35 animals expressing sGFP::ATZ or sGFP::ATMwere sorted into 384-well plates. To minimize variability, onlyPnhx2sGFP::ATZ animals within a tight fluorescence window were sortedinto the wells. Nearly the entire well was imaged in channel 1 usingbrightfield illumination (FIGS. 16A, 16D). These images, which were notused to identify individual animals, simply confirmed that thatcomparable numbers of young adult animals of both lines were sorted intothe wells. Using channel 2 and 3, respectively, SpotDetector identifiedthe red heads (FIGS. 16B, 16E) and either sGFP::ATM (barely detectableat the integration time used, FIG. 16C) or sGFP::ATZ (FIG. 16F)expression in the two different transgenic lines. Next, the red-headsdetected in channel 2 were used to determine a “head count” and to showthat the actual number of animals sorted into each well were nearlyidentical (FIG. 16G). Finally, the images obtained in channel 3 wereused to measure three different parameters in each of the wellscontaining sGFP::ATM or sGFP::ATZ expressing animals (FIGS. 16H-16J).Data analysis indicated that the total GFP intensity, number of GFPspots or GFP area divided by the head count (i.e., parameter average peranimal) were significantly increased in the sGFP::ATZ as compared to thesGFP::ATM expressing animals (FIGS. 16H-16J). Indeed, at the integrationtime used, sGFP::ATM expression was not significantly above that ofwild-type animals.

From these data it was concluded that the steady-state amounts ofsGFP::ATZ in the transgenic line as compared to the control animalsprovided a dynamic range amenable to screening for compounds thataltered sGFP::ATZ accumulation. However, prior to initiating a HCScampaign, the overall quality of the assay, using the Z′-factor as ametric, was tested (87). The Z′-factor, which is calculated from themean and the SD of the negative and positive control populations, is agood indicator of the assay quality, robustness and reproducibility.Values between 0.5 and 1 are considered excellent and necessary beforeembarking upon a HTS/HCS campaign. To determine the quality of thisassay, 180 wells containing wild-type and sGFP::ATZ animals were imagedusing the ArrayScan VTI. In a representative experiment, the mean totalspot area per sGFP::ATM and sGFP::ATZ animals were 0.2±0.7 and 194±22.4,respectively (FIG. 16K). The Z′-factor for this assay was ˜0.7. Within asingle experiment (sort) the Z factor remained constant formplate-to-plate. However, the Z′-factor would vary as much as 0.4 to 0.7from day-to-day depending mostly on the size of the sort-window used toselect the Pnhx2sGFP::ATZ animals.

Compound Screen:

To test the HCS protocol, a pilot drug screen was performed using thelibrary of pharmacologically active compounds (LOPAC 1280™, 1280compounds). Tight gating parameters for total fluorescence were used tosort 35 young adult Pnhx2sGFP::ATZ animals into wells of 384-well platescontaining 50.1m of a LOPAC compound and 0.5% DMSO. Pnhx2sGFP::ATZanimals incubated with 0.5% DMSO served as untreated controls and wereplaced in the first-two and last-two columns of each plate. In arepresentative experiment, plate 1 of the LOPAC library was set-up forscreening on day 1, and 3 other plates were set-up on the next day.After 24 incubation at 25° C., animals were immobilized by the additionof NaAz and placed in the ArrayScan VTI for automated imaging. Toexamine for systematic errors, the raw data (total spot area/animal)were depicted as a plate-well scatter plot (FIG. 17A). From these data,a small amount of drift was seen in plate 1 in comparison to plates 2-4.This difference reflects a wider sort-window used to collect animals onday 1 in comparison to that used on day 2. As the average values of thenegative controls and that of the sample wells were similar, we combinedthe control and samples wells for normalization and to identifypotential hits using the z-score (FIG. 17B). The ArrayScan VTI readsmicrotiter plates by rows, alternating from left-to-right, and thenright-to-left. For some assays, we noted that the control fluorescencevalues would drift upwards slightly in rows towards the bottom of theplate. This drift appeared to correlate with an increase in chambertemperature during the scanning period and was minimized by cooling thechamber with a fan or shortening the read times by using 2.5× objectivewith a 0.6 coupler. Nonetheless, intra-plate variation was controlledfor by presenting the data as a B-score (FIG. 17C). Under the sameconditions, the entire screen was repeated on a single day. An averagerank-score was created for each compound by first compiling a list foreach screen based on ascending B-scores, and then calculating theaverage rank for each compound (TABLE 5). To verify potential hits, wearbitrarily focused on those compounds with rank-scores <110 (n=33)or >1225 (n=15). Generally, compounds with these rank-scores hadB-scores lesser or greater than 3 in at least one of the screens, anddemonstrated the ability to significantly decrease or increase sGFP::ATZaccumulation, respectively. Based on cost and commercial availability,we selected 16 compounds to test for dose-dependent effects (TABLE 5).Cantharidin (FIG. 17D), fluphenazine (FIG. 17E) and pimozide (FIG. 17F)were representative examples of 6 of 12 compounds that showed adose-dependent decrease in sGFP::ATZ accumulation; whereas tyrphostatin(FIG. 17G) was an example of 3 of 4 compounds that showed an increase insGFP::ATZ accumulation. Interestingly, all three compounds thatdecreased GFP::ATZ accumulation were isolated previously in screens forcompounds that enhance autophagy, an known elimination pathway for ATZ(91, 33). When animals expressing the Pnhx-2mCherry::lgg-1 transgenewere treated, the distribution of mCherry::LGG-1 changed from diffuselycytosolic to punctate, suggesting an increase in the number ofautophagosomes (FIG. 18). Taken together, these studies suggest thatthis screening assay was capable of identifying hit compounds thatsignificantly altered sGFP::ATZ accumulation. A dose-response effect forpimozide and fluphenzine is demonstrated in FIG. 19 and FIGS. 20 and 21,respectively.

TABLE 5 Rank- Overall score^(a) rank-order^(b) Potential hit compoundCompounds that decreased ATZ accumulation: 2.0 1 ivermectin^(d) 2.5 2cantharidin^(c) 10.5 3 L-655,240 24.0 4 GR 125487 sulfamate salt 28.0 5muscimol hydrobromide 34.5 6 DL-homatropine hydrobromide 36.5 7L(−)-norepinephrine bitartrate^(d) 41.5 8N-(2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl)- 3-methoxybenzamide 51.0 9cefmetazole sodium^(d) 52.5 10 HA-100 56.0 11 SB 206553 hydrochloride57.5 12 L-701,324 57.5 13 phenamil methanesulfonate^(d) 58.0 14 rolipram61.0 15 doxepin hydrochloride^(d) 61.5 16 beta-chloro-L-alaninehydrochloride 65.0 17 S(−)-UH-301 hydrochloride 72.5 18 L-alpha-methylDOPA 73.5 19 taxol^(c) 74.0 20 cis-(Z)-flupenthixol dihydrochloride 75.521 10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride 79.0 22cantharidic acid^(c) 81.0 23 fluphenazine dihydrochloride^(c) 83.5 24tamoxifen citrate^(c) 85.0 25 indirubin-3′-oxime 89.5 26 (−)-bicucullinemethbromide, 1(S), 9(R) 90.0 27 cephradine 93.0 28 indatralinehydrochloride 95.5 29 5-carboxamidotryptamine maleate 98.0 30 tyrphostinAG 112 103.5 31 prochlorperazine dimaleate 105.5 32 B-HT 933dihydrochloride^(d) 107.5 33 pimozide^(c) Compounds that increased ATZaccumulation: 1263.0 1 GW2974 1256.0 2 thapsigargin^(c) 1255.0 3 SB224289 hydrochloride 1255.0 4 clotrimazole 1253.5 5 IC 261 1245.5 6tetradecylthioacetic acid 1245.0 7 tyrphostin 1 1240.5 8(+)-bromocriptine methanesulfonate 1238.0 9 L-162,313 1236.5 10tyrphostin AG 879^(c) 1236.5 11 IIK7 1234.0 12 glipizide^(d) 1233.5 12WIN 62,577 1231.0 14 (R)-(+)-WIN 55,212-2 mesylate 1229.5 15rottlerin^(c) ^(a)Rank-scores were calculated by averaging compoundrankings based on ascending B-scores from two independent drug screens.Compounds with rank-scores <110 or >1225 significantly decreased orincreased the accumulation of sGFP::ATZ inclusions, respectively.^(b)Overall rank-order, based on relative rank-scores, for compoundsthat decreased or increased sGFP::ATZ accumulation. ^(c)Compounddemonstrated a dose-dependent response. ^(d)Compound failed todemonstrate a dose-dependent response.

7.3 Discussion

Prior to the instant invention, two major obstacles have blocked the useof small animals, such as C. elegans, in high-throughput, high-contentscreening protocols: the absence of 1) a high-quality assays and 2) anautomated system to capture, analyze and store data documenting thebiological effects of thousands of compounds (77). In the experimentsdiscussed herein, in order to improve assay quality, focus was initiallyplaced on parameters that affected sample population variability.Despite using integrated and staged transgenic lines, the fluorescenceintensity of the sGFP::ATZ-expressing animals varied two-fold. Thisvariability was minimized in the assay population by using the COPAS™BIOSORT to collect a precise number of animals using a tightly-gatedfluorescence intensity window. The growth conditions in the microtiterwells also had a profound affect on assay quality. C. elegansMaintenance Medium (a chemically defined, bacteria free medium) appearedto be an ideal growth medium for animals cultivated in microtiterplates, but intense autofluorescence precluded further use (88).Ultimately, S Medium supplemented with antibiotics and E. coli (OP50)was used. Antibiotics were included to limit bacterial growth, which hada tendency to overgrow the cultures and kill the nematodes. Defining theoptimal growth conditions, which vary depending on the length of theassay period and the number and condition of the animals, was importantto developing a robust and reproducible assay.

The second major impediment to the routine use of C. elegans in HCS wasthe lack of systems that automated the time-consuming process of imageacquisition, analysis and storage. This bottleneck is evident in thefirst series of relatively low-throughput and labor-intensive C. elegansdrug screens (67, 68, 72, 89, 90). Compound effects were assessed bydirect inspection of animals in microtiter plate wells using astereomicroscope or of images captured by a CCD camera. Althoughsensitive for the detection of certain phenotypes, such as alterationsin movement or morphology, manual inspection of plates or images is timeconsuming and tedious for HTS campaigns scaled for assayinghundreds-of-thousands of compounds (67, 68, 72). Moreover, operatorfatigue increases variability and decreases specificity. An enzymaticassay that measures fluorescent substrate conversion in culture mediacan be automated, but the effects of compounds on the whole animal arelost (89). Recently, Moy et al., reported an automated high-throughputscreen for novel antimicrobial compounds that protect C. elegans from alethal dose of S. faecalis (70). While their automated screening assaywas five-times faster than screening manually, the algorithm was limitedto a simple yes-no (live-dead) assessment and was unable to the effectsof compounds on individual animals. Taken together and as compared toestablished cell-based HCS protocols, these studies suggest that wholeanimal HCS was cumbersome and lacked the refinements in imageacquisition and analysis to quantitatively assess compound effects oncontinuous physiological variables such as growth and developmentautophagy, misfolded protein disposition and cell permeability. Incontrast, the HCS format of the invention could capture 5-channelmultiparametric images of each well of microtiter plate using anautomated inverted fluorescence microscope and image analysis software.Since the images were stored on a server, different algorithms could beapplied at different times to extract various quantitative measures,such as fluorescent spot count, spot area or spot intensity per animal.These types of qualitative measures could never be assessed accuratelymanually, as the time required to count, for example, a dozenfluorescent spots in 35 animals in each well of a 384-well plate wouldrapidly fatigue even the most fastidious observer. In addition, imagingsystem use for data acquisition affected assay quality, which wasimproved by optimizing the microscope's autofocus and scanning times,and the degree of magnification used to scan the wells of 96- versus384-well plates. The analysis algorithms, which control the fluorescenceintensity cut-offs used to define fluorescent objects and must beempirically defined for each marker system, also had a significantimpact on overall assay quality. By manipulating these parameters,Z′-factors were consistently obtained, which serves as a measure ofassay quality, in the excellent range of 0.5 to 0.7, scores that rivaledthose of the highest quality cell- or molecule-based HTS schemes (87).Based on these studies using transgenic animals expressing misfoldedsGFP::ATZ, the ArrayScan VTI and the BioApplication programs possess theautomation, speed and sensitivity to generate a high-quality assay thatwould permit the quantitative assessment of compound libraries on acontinuous physiological variable, such as misfolded proteinaccumulation. Moreover, by optimizing the imaging analysis, the scanningtime of a 384-well plate was reduced to ˜30 minutes. Thus, ˜6,000compounds could be screened in a typical workday, or ˜18,000 compoundsper day if the ArrayScan VTI were configured with an automated plateloader. Screening using of compound effects on a discrete variable, suchas a live-dead screen, would be even faster.

As a test of this C. elegans screening strategy, a limited drug screenwas performed for compounds that affect accumulation of the misfoldedhuman serpin, α1-antitrypsin (sGFP::ATZ). Of the 6 compounds inducing adose-dependant decrease in sGFP::ATZ accumulation, four (cantharidin,tamoxifen, fluphenazine and pimozide) belong to classes of drugs thatwere identified previously as enhancers of autophagy (91, 33)—aphysiological process involved in the elimination of ATZ (30). Thus, thedrug discovery strategy outlined herein has significant clinical import,as C. elegans has been used to model several protein misfoldingdisorders including Alzheimer's disease (92) and polyglutamine repeatdisorders (93). Accordingly, live animal HCS for compounds thatameliorate disorders of proteostasis is feasible (94).

8. EXAMPLE Fluphenazine Reduces ATZ in Human Cells

Both fluphenazine and pimozide have a dose-dependent effect on GFP+inclusions in ATZ worms with almost complete elimination of theinclusions at 10 μM. These drugs also appear to be effective in reducingATZ levels in the mammalian cell line model of AT deficiency, HTO/Z. Anexample for the effect of fluphenazine in HTO/Z cells is shown in FIG.22. The cells were incubated for 48 hrs with fluphenazine in severaldifferent concentrations and then subjected to Western blot analysis forAT. The results show that fluphenazine mediates a dose-dependentdecrease in insoluble ATZ levels. The effect is evident at doses of 1-10μM. Interestingly this drug appears to lead to an increase in solubleATZ with no significant change in levels of the control GAPDH. Thesedata imply that the drug solely affects autophagic disposal of insolubleATZ and that leads to more soluble ATZ. This is different from CBZ whichappears to affect autophagic as well as proteasomal degradation.

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Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

We claim:
 1. A method of treating a clinical disorder associated withincreased protein aggregation comprising administering, to a subject inneed of such treatment, an effective amount of pimozide or astructurally related compound.
 2. The method of claim 1 where thedisorder is α1-antitrypsin deficiency.
 3. The method of claim 1, wherethe disorder is selected from the group consisting of Alzheimer'sDisease, Parkinson's Disease, amyloidosis, Huntington's disease andprion diseases.
 4. A method of treating a clinical disorder associatedwith increased protein aggregation comprising administering, to asubject in need of such treatment, an effective amount of fluphenazineor a structurally related compound.
 5. The method of claim 4 where thedisorder is α1-antitrypsin deficiency.
 6. The method of claim 4, wherethe disorder is selected from the group consisting of Alzheimer'sDisease, Parkinson's Disease, amyloidosis, Huntington's disease andprion diseases.
 7. A method of treating a clinical disorder associatedwith increased protein aggregation comprising administering, to asubject in need of such treatment, an effective amount of tamoxifen or astructurally related compound.
 8. The method of claim 7 where thedisorder is α1-antitrypsin deficiency.
 9. The method of claim 7, wherethe disorder is selected from the group consisting of Alzheimer'sDisease, Parkinson's Disease, amyloidosis, Huntington's disease andprion diseases.
 10. A method of treating a clinical disorder associatedwith increased protein aggregation comprising administering, to asubject in need of such treatment, an effective amount of taxol or astructurally related compound.
 11. The method of claim 10 where thedisorder is α1-antitrypsin deficiency.
 12. The method of claim 10, wherethe disorder is selected from the group consisting of Alzheimer'sDisease, Parkinson's Disease, amyloidosis, Huntington's disease andprion diseases.
 13. A method of treating a clinical disorder associatedwith increased protein aggregation comprising administering, to asubject in need of such treatment, an effective amount of cantharidin,cantharidic acid, a salt thereof or a structurally related compound. 14.The method of claim 13 where the disorder is α1-antitrypsin deficiency.15. The method of claim 13, where the disorder is selected from thegroup consisting of Alzheimer's Disease, Parkinson's Disease,amyloidosis, Huntington's disease and prion diseases.
 16. A method oftreating a clinical disorder associated with increased proteinaggregation comprising administering, to a subject in need of suchtreatment, an effective amount of a compound listed in TABLE 4 or TABLE5 herein.
 17. An isolated nucleic acid comprising a nucleic acidencoding a human protein with a tendency to aggregate or a polymerizableportion thereof operably linked to a Caenorhabditis elegans promoter,where said nucleic acid encoding a human protein with a tendency toaggregate or a polymerizable portion thereof comprises one or moresynthetic intron.
 18. The nucleic acid of claim 17, where the protein isselected from the group consisting of ATZ, huntingtin, synuclein,amyloid beta, neuroserpin, ubiquitin, neurofilament protein, alpha Bcrystallin and tau, where all or a polymerizable portion thereof iscomprised in a fusion protein with a first fluorescent protein.
 19. Thenucleic acid of claim 17 where the Caenorhabditis elegans promoter isselected from the group consisting of a neuron-specific promoter, agut-specific promoter, a muscle-specific promoter, a pharynx-specificpromoter and a tail-specific promoter.
 20. A Caenorhabditis eleganscarrying a transgene comprising the nucleic acid of claim
 19. 21. TheCaenorhabditis elegans of claim 20, further carrying a transgenecomprising a marker construct comprising a marker gene encoding a markerprotein operably linked to a second Caenorhabditis elegans promoter, theexpression of which results in a single detectable region per worm. 22.The Caenorhabditis elegans of claim 21, where the marker protein is asecond fluorescent protein.
 23. The Caenorhabditis elegans of claim 21,where the second Caenorhabditis elegans promoter is selected from thegroup consisting of a pharynx-specific promoter and a tail-specificpromoter.
 24. A model system for α1-antitrypsin deficiency comprising aCaenorhabditis elegans carrying a transgene comprising a nucleic acidencoding an α1-antitrypsin mutant selected from the group consisting ofATZ, Siiyama and Mmalton comprised in a fusion protein with afluorescent protein, operably linked to a C. elegans promoter, wheresaid promoter is a gut specific promoter, and where said nucleic acidencoding ATZ comprises one or more synthetic intron.
 25. The modelsystem of claim 24, where said Caenorhabditis elegans further carries atransgene comprising a marker gene encoding a marker protein, operablylinked to a Caenorhabditis elegans promoter.
 26. A model system for adisorder associated with protein aggregation in neurons comprising aCaenorhabditis elegans carrying a transgene comprising a nucleic acidencoding a protein with a tendency to aggregate, selected from the groupconsisting of human huntingtin, synuclein, amyloid beta, neuroserpin,ubiquitin, neurofilament protein, alpha B crystallin, tau and anaggregatable portion of any of the foregoing human proteins, comprisedin a fusion protein with a fluorescent protein, operably linked to a C.elegans promoter, where said promoter is selected from the groupconsisting of a neuron-specific promoter, a gut specific promoter, amuscle specific promoter, or a pharynx-specific promoter, and where saidnucleic acid encoding a protein with a tendency to aggregate comprisesone or more synthetic intron.
 27. The model system of claim 26, wheresaid Caenorhabditis elegans further carries a second transgenecomprising a marker gene encoding a marker protein, operably linked to asecond Caenorhabditis elegans promoter.
 28. The model system of claim26, where the protein with a tendency to aggregate is human amyloidbeta, or a polymerizable portion thereof, and the system is a model forAlzheimer's Disease.
 29. The model system of claim 26 where the proteinwith a tendency to aggregate is human synuclein, or an aggregatableportion thereof, and the system is a model for Parkinson's Disease. 30.The model system of claim 26 where the protein with a tendency toaggregate is human huntingtin, or an aggregatable portion thereof, andthe system is a model for Huntington's Disease.
 31. The model system ofclaim 26 where the protein with a tendency to aggregate is human tauprotein, or an aggregatable portion thereof, and the system is a modelfor chronic traumatic brain injury.
 32. The model system of claim 26where the protein with a tendency to aggregate is human mutantneuroserpin, or an aggregatable portion thereof, and the system is amodel for Familial encephalopathy with neuroserpin inclusion bodies. 33.A method of determining whether a test compound has activity in treatinga disorder of protein aggregation, comprising: (i) administering saidtest compound to a plurality of transgenic Caenorhabditis eleganscarrying (a) a first transgene comprising a nucleic acid encoding ahuman protein with a tendency to aggregate, or an aggregatable portionthereof, operably linked to a Caenorhabditis elegans promoter, where theexpression of the protein results in a detectable accumulation of theprotein in the Caenorhabditis elegans and (b) a second transgenecomprising a marker construct comprising a marker gene encoding a markerprotein operably linked to a Caenorhabditis elegans promoter; (ii)determining the change in the amount of human protein or aggregatableportion thereof associated with the administration of test compound inthe plurality of Caenorhabditis elegans; (iii) using the marker protein,determining the number of Caenorhabditis elegans in said plurality; (iv)using the results of (ii) and (iii), determining the change in theamount of human protein per worm resulting from the administration oftest compound; wherein, if administration of the test compound resultsin a significant decrease in the amount of human protein per worm, thenthe test compound is indicated to be therapeutically effective in adisorder of protein aggregation.
 34. The method of claim 33, where thehuman protein with a tendency to aggregate, or an aggregatable portionthereof, is comprised in a fusion protein together with a firstfluorescent protein.
 35. The method of claim 33, where the markerprotein is a second fluorescent protein.
 36. The method of claim 33,where the first fluorescent protein and the second fluorescent proteinare not the same.
 37. The method of claim 33, where the expression ofthe marker protein results in a single detectable region per worm.